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Hello, I would like to ask from everyone's perspective what is the biological relevance and impact if the neurons that are being affected by an exogenous stimulus is (1) peptidergic or non-peptidergic neuron, (2) and their respective class of nerve fibers?
Currently, I am still consolidating and distinguishing these concepts because I think these are important research questions in molecular and cellular neuroscience projects.
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If these are people, then a clinical response to the administration of naloxone is likely. If the experiment... is a microelectrode neuronal response also using blockade of opiate receptors.
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Dear scientists,
I have a question regarding self-renewal assay.
Instead of manually counting neurospheres in each well after seeding at clonal density
is it possible that counting is done automatically (for example with FACS)?
Have anybody tried it?
Thanks a lot for your answers and help.
Regards, Snjezana
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Also do keep in mind, I guess depending on how often you do these counts, that there are more automated options as well. The white paper below is for mammospheres but should translate to neutrospheres as well.
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I am starting to use BiC/4AP for an experiment to stimulate hippocampal neurons. This technique has been used previously by other labs and a former member from my own lab. I have tried several times, but cannot seem to get the same results as others. I am using bicuculline and 4AP from at least 10 years ago that has been stored at room temperature in a dessicant box. The bicuculline is stored in aluminum foil also to prevent light exposure.
I'm wondering if my experiment is not working because the drugs are too old. I have tried to look for the shelf life of the drugs but cannot find much. Does anybody have experience working with these drugs and have any idea of how long they are good for when stored at room temperature?
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Long term storage of bicuculline should be at -20 oC and this will only be useable for approximately 1-2 years. 4-AP should also be stored at -20 oC for long term and will only be useable for approximately 6-12 months.
Therefore, you should discard the old solutions and order fresh products.
(An tip for identifying shelf life is to look at the stability and storage section in the product information sheet for each product)
Hope this helps!
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I'm looking to collaborate.
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I am Assistant Professor at Department of Zoology, Goa University, Goa, India. Along with my teaching assignment, I do my research in the area of developmental neurogenesis using chick embryos and making an attempt to move ahead with combination of signal transduction involving neurotransmitters and neuromodulators during developmental neurogenesis. Had also worked with rat pups in past. Do not have full fledged neuroscience lab but having animal cell culture facility and enough requirement of lab to sustain work in developmental neurogenesis.
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My lab is studying neuroprotective effect towward Sh-SY5Y cells. However, when I seed the Sh-Sy5Y cells in Sigma 96 well plate coated with PDL (cls3842), 3 days later, all Sh-SY5Y cells clumped together forming small lumps. In normal, they should be divided indivudally, equally spread inside the wells. This abnormal phenomenon does not appear when we used in a 96 well plate without PDL.
It surprises me since I read papers that using SH-SY5Y cells coated with PDL before.
I am wondering have you heard of any similar observation, and do you have any suggestion to prevent this?
Or do you guys have any suggestion for how to do pdl toward plates?
Thanks!
Thomas
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Hi Di, use the HistoGrip for SH-SY5Y differentiation is super good. https://www.thermofisher.com/order/catalog/product/008050#/008050.
HAVE A TRY.
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If so, has the use of the GloChicks been more effective than using a non-transgenic chicken, in terms of imaging quality?
Thank you.
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We would like to electroporate neurons in organotypic slice cultures with DNA for genetically-encoded flourescent reporters (pH reporters, GEVIs etc). We would like to use an anionic dye that can help us visualize the electroporation but won't hang around in the cell so long that it would interfere with the later visualisation of the expression of the genetically-encoded flourescent reporter several days later. We worry that if we use something like a Alexa-flour 488 Dextran this will hang around so long that it would interefer with later flouresence measurements from the genetically encoded reporters. Any suggestions would be greatly appreciated.
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We do a lot of single-cell electroporation and routinely include one of the non-Dextran Alexa dyes - 488 or 564 usually. Never seemed to have any problem with the fluorophore. Probably the Dextran dye also won't cause any problems but I never tried it...
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would like to know how to differentiate them and how are the phenotypic changes? do they give rise to dopaminergic or glutamanergic phenotypes?
what would be the agents to induce differentiation in these cells?
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Hi Abu Bakkar Siddik, I would try differentiation for 48hrs up to 96 hrs even. Also good differentiation markers include Synaptophysin, NeuN, and PSD95
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We are plating primary hippocampal neurons from rats at post-natal day 0-2.  At plating 350,000 cells/mL with a total of 4 mls/plate.  We coat our plates with PDL and then with lignin.  At plating, the cells are placed in MEM + FBS + L-glutamine + Pen/Strep.  The day after plating they are switched to what we call neuron media containing Neurobasal A + B27 + Glutamax + FdUR + Uridine+ Pen/Strep.  We feed the plates every 3-4 days by removing 2 mls and replacing 2 mls of fresh neuron media.  The cells look great all up to the change right around 14 days.  After this change, like clockwork, the cells start dying the next day.  I make up the fresh media the day of changing and do not let it sit for more than 10 minutes in the waterbath.  Any suggestions why the cells do well for so long and then start dying at this last media change?  We are hoping to work with more mature neurons (day 14 or older) but so far have not been able to. 
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Nice Contribution Margarethe Bittins
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I am curious about two specific things:
- Why do pseudounipolar neurons have one axon (as opposed to a dendrite + axon like multipolar neurons)? How does this structure reflect sensory function?
- How do potentials propagate through the axon? Since there is no axon hillock for summation, does that mean no summation occurs? Is there still a threshold potential that needs to be met? Or does every graded potential get transmitted through the axon?
Can someone familiar with any of these questions help out or provide a resource I can refer to?
Thank you!
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Hi Sehej
Pseudounipolar pattern of sensory neurons acts as low-pass filtering, potentially regulating sensory information reaching the spinal cord. Thus, by impedance mismatch between membrane point in the vicinity of T-junction, this has been recognized as a site where spike propagation may fail.
As for action potential propagation through the axon, this is a more broad question. Actually, in a really simplistic summary, sensory neurons have "transductors" (specialized proteins that converts physical energy - thermal, mechanic, chemical - into electrical signal) in their peripheral end; these transductors creates a "generator potential", according to specifical thresholds. If, and only if, these generator potentials reach some specific area in the membrane with a larger density in voltage gated channels (as Na channels) within these second step threshold, an action potential (spike) is generated and conveyed until the "T-junction" filter described above.
Some references for better comprehension
1 -Al-Basha, Dhekra, and Steven A. Prescott. "Intermittent Failure of Spike Propagation in Primary Afferent Neurons during Tactile Stimulation." Journal of Neuroscience 39.50 (2019): 9927-9939.
2 - Sundt, Danielle, Nikita Gamper, and David B. Jaffe. "Spike propagation through the dorsal root ganglia in an unmyelinated sensory neuron: a modeling study." Journal of neurophysiology 114.6 (2015): 3140-3153.
Regards,
Tiago Avelar
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Working on drosophila adult brains here, I want to look at the "average" activity of a neuronal population. I am not interested in their response to a stimuli but rather at their spontaneous firing. 
Will PFA fixation of GCaMP expressing cells give me this information? 
I can not find any paper where GCaMP is fixed so maybe there is a reason that it doesn't work that I am not aware of. 
Thank you all!
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Liubov, see my answer below: if you prepare the fixed sample appropriately, there is enough native fluorescence so no staining is required.
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Hei,
I want to analyze apoptosis using AnnexinV/ PI. I am working with various neuroblastoma cell lines such as SKNAS, SHSY5Y SKNBE(2), Kelly and several other.
I am using the FITC-AnnexinV/PI kit from BD.
The assay worked always fine when I analyzed apoptosis in SKNAS.
However, when I used the same protocol for SKNBE(2), I always got approximately 80% Annexin potitive cells. And these cells were not treated- thus, these cells were healthy cells that should not have more than 5-10% apoptotic cells.
Today, I analyzed apoptosis of Kelly and SHSY5Y cells. Here, I also got 70-80% Annexin positive cells in untreated cells.
Might the cell membrane of these cell lines have phosphatidylserine in the outer leaflet of the plasma membrane even if cells are not apoptotic?
If so, the assay would not work for these cell lines...Did you have similar problems when using this assay or read about it?
This is my protocol:
  1. Transfer medium to 1.5 ml tubes
  2. Wash with 300 μl PBS and transfer PBS to 1.5 ml tubes
  3. Add 200 μl trypsin and incubate 3 minutes
  4. Use the medium to inactivate trypsin
  5. Transfer the cell suspension back to 1,5 ml tubes
  6. Centrifuge at 200 g 5 minutes
  7. Resuspend cells in 500 μl PBS (2 wells are merged)
  8. Centrifuge at 200 g 5 minutes
  9. Resuspend in 100 μl Annexin V binding buffer
  10. Add 5μl FITC-Annexin V and PI
  11. Gently vortex cells and incubate 15 minutes at RT in the dark
  12. Add 400 μl 1x binding buffer to each tube
  13. Analyze by flow cytometry
PI:  Laser 561 nm; Filter 670/30
AnnexinV: Laser 488nm; Filter 530/30
Controls:
  • unstained cells (to set gates)
  • PI only
  • AnnexinV only
  • PI/AnnexinV
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Hi Sarah,
I am also facing the same problem with Neuro 2A (Mouse Neuroblastoma) cell line. In healthy cell, I am also getting 60-70% cell viability. To troubleshoot this, initially, I ran the cell with 10, 15 and 20 % FBS and got 70% (Max) cell viability in 15% FBS Cells. Then after used gradient of PI (3, 5 and 10 μl ) and annexin but still got the same result. Kindly suggest to me If you have figured out this problem. Thank you in advance
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We are making whole-cell patch clamp recordings from mouse (and human) fast-spiking interneurons using Axopatch 200B amplifiers. We see a sharp overshoot after action potentials (see red trace in the image) which we assume is an artefact caused by pipette capacitance correction? Could anyone confirm this? What is best practice when making current clamp recordings using a Axopatch 200B? Should one use both pipette capacitance correction and 100% series resistance correction? We are trying to characterise the intrinsic properties of the neurons but it seems like pipette capacitance correction is making a huge difference. Any help would be much appreciated.
Currently we do the following before begining our recordings:
At the moment we do this: 
1) in cell attached mode we use pipette capacitance correction to remove capacitve transients
2) we break through into whole cell mode
3) we run a short current step and find the correct series resistance of the cell with 100% correction
We then perform our recordings (such as current steps)
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Hi Joseph
You are doing the right things and also getting good advise as above (Miroslav N Nenov).
But... I would suggest that if you have a bridge amplifier in the lab (eg. Axoclamp 2B) that you use that instead of the 200B. Patch clamp amplifiers have the potential to introduce some distortions to fast waveforms, so if you have a bridge amplifier you will probably be better off.
see this paper:
And I think that with Multiclamp this is not a problem anymore.
All the best
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I'm following this protocol but the TEER that I get is very low as compared to theirs in the paper. My cells are not as confluent too although i seed the same amount of cells.
Does anyone know a good method to increase TEER?
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Our company develops 3D cell barrier culture systems for in vitro study of virtually any cell barrier, and the system comes equipped with both a Fluid Perfusion Unit and a Trans-Endothelial/Epithelial Electrical Resistance (TEER) Measurement Unit! The use of a vessel-shaped 3D environment has been proven more effective than 2D systems like Transwell (
Article Santaguida S, Janigro D, Hossain M, Oby E, Rapp E, Cucullo L...
), and our advanced TEER Measurement Unit allows for frequency sweeps between 0.1-1,000 Hz, automated time point sampling, logging of data to Excel, and additional measurement of phase angle for cell capacitance calculations! For those that are committed to Transwell use, we also have a TEER Measurement Unit that is compatible with nearly all Transwell products (Endohm cell cup chamber, STX2 "chopstick" electrodes, etc) and allows the user to greatly expand their testing capabilities with Transwell equipment. Additionally, we have smaller modular systems that connect to microscope-friendly cell culture modules: i.e., a miniaturized 3D cell culture system that you can use right under your microscope! If you or anybody on this thread is interested in learning more, I encourage you to visit www.flocel.com or email me at djanigro@flocel.com, thank you!
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I have read everything from 1mM to 200-300uM to 10uM as far as glutamate concentration.  Some refer to 200-300uM as the "saturation point for glutamate".  
As far as the stock is concerned, we would like to be able to make a concentrated stock and treat with that instead of replacing the media for each treatment well.  Sigma glutamate (https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/g1251pis.pdf) has a solubility limit of 8.6mg/mL in water, while sigma monosodium glutamate (https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/g1626pis.pdf) is soluble up to 100mg/mL.  The solubility of MSG is preferable, but we will likely have to pH this.  Would the extra sodium ions be a worry?
Thanks in advance!
Connor
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try first on a 24h period and if you get a good toxicity with 100 µM glutamate you may thus test a time course: 30 min, 60 min, 1, 2, 6, h to test the optimal period of treatment under your conditions. If you use high amounts of glutamate, such as 5 or 10 mM, as tested by some authors, you may possibly kill your cell by an other mechanism such as inhibition of transporter xc-, which exchange glutamate against cysteine, thus leading to an oxidative shock (so called oxytosis).
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I am hoping to perform biolistic transfection of organotypic brain slice cultures. In my previous lab we used the Biorad Helios Gene Gun System - which effectively is just a pressurized gun that fires gold bullets through nylon tubing. However the system costs $30 000 which is exorbitant for the technology! Has anyone used alternatives such as this Chinese competitor: http://www.scientzbio.com/gene-transduction-device/portable-gene-gun-sj-500.html
Any thoughts would be welcome!
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Hi,
I have not heard of that company personally but that doesn't mean anything.
I have however seen multiple labs succeed in building their own gene gun from scratch.
Attached is a reference that may help you get started if you choose to go that way.
And here is an easier to follow web article entirely on the topic of a homemade gene gun.
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I am studying the establishment of synapsis in N2a cells, comparing synapsis protein expression in differentiated and non-differentiated N2a cells.
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Hello Perez
I differentiate N2a Cells by exposing them to 10 uM Retinoic Acid in 2.5% FBS DMEMF12 Medium for 8 days. You will get fully differentiated neurons after 8 days of differentiation.
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We are looking to reconstruct biocytin filled neurons from confocal image stacks. I realise Neurolucida is the gold standard but even the Neurolucida 360 lite version is hideously expensive (~$15 000). Is Neuronstudio a viable alternative despite not being updated since 2009. Are their suitable plugins for Fiji? It would be really great to reconstruct in 3D. Any thoughts would be appreciated.
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For reconstructions from 3D image stacks, we have been quite satisfied with the Neuromantic freeware:
It does what it is supposed to, with no bells and whistle that complicate matters more than they help. With Neuromantic, I have put dozens of undergraduate students on the manual reconstruction work, they get their lab experience, we get our reconstructions, everybody's happy. We've done >150 reconstructions in the lab this way. Works great, but support is nil and updates rare.
I have heard good things about Fiji/Simple Neurite Tracer but never tried it. There is also Herman Cuntz's TREES toolbox (http://www.treestoolbox.org), which is more advanced. Finally, you might benefit from the option not to reconstruct at all (Ferreira et al: Neuronal morphometry directly from bitmap images. Nature Methods 2014 DOI:10.1038/nmeth.3125.).
All of this requires 3D image stacks though, so you do not work "live" like people often do with Neurolucida. However, working offline comes with advantages too.
For our work with Neuromantic, see below, in particular Blackman et al:
Blackman AV, Grabuschnig S, Legenstein R, & Sjöström PJ: A comparison of manual neuronal reconstruction from biocytin histology or 2-photon imaging: morphometry and computer modeling. Frontiers in Neuroanatomy (2014) 8:65, DOI: 10.3389/fnana.2014.00065.
Lalanne T, Abrahamsson T, & Sjöström PJ: Using Multiple Whole-Cell Recordings to Study Spike-Timing-Dependent Plasticity in Acute Neocortical Slices. Cold Spring Harb Protoc (2016) 10.1101/pdb.prot091306
Buchanan KA, Blackman AV, Moreau AW, Elgar D, Costa RP, Lalanne T, Tudor Jones AA, Oyrer J, & Sjöström PJ: Target-Specific Expression of Presynaptic NMDA Receptors in Neocortical Microcircuits. Neuron (2012) 75:451-466.
Lalanne T, Oyrer J, Mancino A, Gregor E, Chung A, Huynh L, Burwell S, Maheux J, Farrant M, and Sjöström PJ: Synapse-specific expression of calcium-permeable AMPA receptors in neocortical layer 5. The Journal of Physiology (2016) 594(4):837-861.
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Hi dear colleagues I need to record AMPA and NMDA currents of CA1 neurons in p21-p32 mice but I do not know what internal solution to prepare. There are papers that use CsCl, other Cs-Glu and other K-Gluc and differ in the use of QX314 (besides that they use different reactants). I really do not know what internal solution is better for this type of records and I am starting in the patch-clamp world. Also, is it possible that you can recommend a publication that supports the use of your internal solution? What care should I have when preparing the internal solution (ATP / GTP) and during the electrophysiological record? I appreciate your help very much
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Hello!
For voltage-clamp recordings the Cs-Methanesulfonate based solution is used in our lab.
127 CsMeS, 10 NaCl, 5 EGTA, 10 HEPES, 6 QX314, 4 ATP-Mg, and 0.3 GTP; pH adjusted to 7.25 with CsOH
It allows to record synaptic currents in pyramidal neurons in slices for prolonged period of time. As it contains the blockers of potassium and sodium channels, the input resistance in whole-cell configuration is rather high (about 250-400 MOhm). I would recommend to voltage-clamp the cell at -50 - -20 mV most of the time during the experiment. Prolonged recordings at more negative voltages decrease the cell viability during the recording. This solution can't be used for current-clamp recordings.
For current-clamp recordings the K-Gluconate based solution is used:
135 K-gluconate, 10 NaCl, 5 EGTA, 10 HEPES, 4 ATP-Mg, and 0.3 GTP (with pH adjusted to 7.25 with KOH)
It allows to record membrane voltage and action potentials in pyramidal neurons in slices. However it does not perform too well in voltage-clamp mode, as the input resistance in whole-cell configuration is quite low (60-180 MOhm depending on cell type). If you use it in voltage-clamp recordings I would recommend to clamp the cell at -90 - -60 mV most of the time during the experiment.
Both of the solutions are prepared in the same manner. First we dissolve all the components in water, except ATP and GTP. After that we rougly adjust pH. Then we rapidly add ATP and GTP, make a final adjustment of pH (ATP decreases the pH) and freeze the solution in 1 ml tubes. The osmolarity of the resulting solutions is about 300 mOsm (for better patching it should be a little lower than the osmolarity of the extracellular solution).
Good luck with your experiments.
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Cells not easy to transfect
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Did you ever try transfecting primary motor neurons with AMAXA?
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Hi all,
I recently begin to learn single-unit recording in the primary visual cortex of mouse brain using tungsten electrodes. Sometimes (but not very often), there is significant bleeding during when I try to remove the skull and the meninges, but in all the cases the vessels eventually stopped to bleed. I wish to know how will bleeding affect neurons nearby? Will neurons die because they have less O2 and glucose supplied?
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Dear Zhou,
That will depend on the level of tissue and bleeding. If there is significant level of blood supply to the area that you are recording from, the reduction in local oxygen supply can cell death. Another factor is the swelling associated with damage to the Pia mater and damage other tissue ( interstitial) damage. Usually as you go down with your electrode you should be able to see/hear (from amplifier) discharges of damaged neurons. If your electrode tip is not blocked and you hear/see no discharges at all. That is bad news, meaning substantial damage. That is what I used to do in my studies, for an example see below.
Best wishes,
Refik
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Most papers talk about co release of neurotransmitters for example...PAG glutamatergic neurons co-release substance P, serotonin, opiods etc....I was wondering if anyone came across a paper showing co-labelling for VGLUT and GAD
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Hi Srinivasa,
The short answer is yes. As an example out of my mind are VTA neurons (http://www.nature.com/neuro/journal/v17/n11/full/nn.3823.html) but I believe they are not the only ones - need to do some literature on this.
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Hi,
I am looking for a good mouse anti-Iba1 antibody for double staining on fixed mouse tissue. I have stained mouse brain microglia with anti-Iba1 from Abcam , used titrations but the double labeling is a issue since I am getting a lot of background or no staining of cell morphology at all.
Does someone has any experience with this antibody or any suggestion to get the best result?
Thank you!
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Hi Lorena,
the rabbit anti Iba1 (1/250) from Wako Pure Chemical Industries, Ltd, Tokyo, Japan) works well.
Good luck.
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Hi! 
Please let me know if you or your lab in Europe have  Ndnf-IRES2-dgCre-D transgenic mice. I will be extremely grateful!
Many thanks!
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We do have this line and currently characterizing it.  This is the line from Allen brain institute.
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We are trying perform patch clamp (whole cell) recordings over basal ganglia neurons (striatum and substantia nigra) from organotypic rat silices that have been cultured between one and two weeks. The issue is that, even when the aspect and morphology of the recorded neurons seem to be normal and healty, after achieving a good seal and acces to the cell the neurons did not present action potentials in response to depolarising current steps. Also, neuronal resistances tended to be higher than in normal BG neurons, in several cases over 1Gohm.
We are using ACSF of 300-310 mOsm and internal patch solution of 290 mOsm. The osmolarity of the culture medium was 280 mOsm and yesterday we have adjusted to 310 mOsm for the new group of slices.
Did you think that by adjusting de osmolarity of the medium will resolve the problem or what else can be done?
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Thanks William, Sheng-Nan and Brandon for your kind replies. I was trying to have some new recordings from the new slices with the change in osmolarity but the history is the same.
William: I´m afraid to agree with you, something terrible is must be happening in some point of the culturing. I will post more information about the growing media and pictures of the current traces as soon as I can hopping to read your replies.
Sheng-Nan Wu: I agree with you, in some cases I’m not sure if have a good access to the cells, but even when I could be on cell attach mode, I don´t see action currents at all. In voltage clamp mode sometimes it looks like the cell it could be closed, but when I switch to current clamp I can see Taus of more than 50 ms then I suppose that I have access to the cytoplasm.
Brandon: Im using holding voltage of 80 or 70 mV and the membrane potentials are variable from -75 to -45. Surely I´ll reply soon with I-V curves to hear about the observations that you can give me. I´m not quite sure that the neurons are making new interconnections between each because the only ones that I have successfully record have just one week and the histology is not yet done, but as far as I have seen, I haven´t notice spontaneous synaptic currents.  Also, I haven´t tried any drug, which one would you recommend.
Thank’s again for your help
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I am thinking on using NeuN? Any comments, suggestions?
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Neuron-specific Class III β-tubulin (TuJ1) is present in newly generated immature postmitotic neurons..so TuJ1 would be best marker for newborn neurons where as NeuN and MAP-2 is for different developmental stages. 
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Is there something specific about neuronal membrane composition at the molecular level that makes it different from other cells membrane?
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I agree that actually there is not much material about the neuron membrane composition, which is something that would be interesting to know; for instance, it could be really useful to unravel the exact mechanism through which atmospheric gas are able to enter neurons and/or to modulate their activity. Todays attention is mostly focused on receptors, but structural elements may play a role in that, beside proteic receptors. If anybody has some material, please let me know! thanks,
Mattia 
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Hi. Recently I've tried to record field potential in brain slice but failed. I use bipolar or monopolar stimulating electrode. Amp is Axopatch 1-D and headstage is CV-4. Recording was done at I-clamp mode. When the recording electrode containing normal ACSF touched the surface of brain slice, I started current injection but I could not see any responses but stimulus artifact. Would you please give me some advice?
As I had not recorded field potential before, I used brain stem slice (I have many experiences here). Age of mice is around P(postnatal day)3~P9. It is MNTB-LSO synapses at pons level.
Stimulating electrode(bipolar or unipolar) was located at MNTB and recording electrode (3~5 megaohm) was at LSO. The resistance of stimulating electrode was also in the same range of that of recording electrode (in case of unipolar electrode). I also tried bipolar electrode but failed.
LSO cells viewed with high magnification were alive and healthy. In voltage clamp mode, postsynaptic currents were elicited by stimulation at MNTB.
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Hi Seungcheol Ahn,
Thanks for the update. That is a non-trivial slice, and one that I don't have personal experience with. My worry would be that you have cut the axons between the MNTB and the LSO.
If I were you, I would cut hippocampal slices. Pretty much any slice you cut that has a complete hippocampus in it will have a connection between CA3 and CA1. If you stimulate in stratum radiatum and put a recording electrode in the pyramidal cell layer you should see a large population spike (> 1mV). If you can not see this, then there is a problem with your stimulating or recording system.
However, if I had to make a guess, other than the worry that you have cut the MNTB LSO axons, it might be that the resistance of your stimulating electrode is too high. If we say that it has a resistance of 5MOhm, and you want to pass 100uA of stimulating current, then that would require 500 Volts, which is far more than any stimulating system that I know of has available. You'd be limited to less than 20 uA, which is starting to get pretty low (not that that amount can't work, it's just not what I would recommend for testing a system).
Try stimulating with a simple Tungsten wire (or something similar), with a resistance of 100kOhm or so.
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 Has anyone used them? (https://www.abmgood.com/productSearch.php?searchQuery=T0251) I have been in contact with the authors on the cited paper (http://www.ncbi.nlm.nih.gov/pubmed/25561230) but I would appreciate any other experiences with them. The company cannot provide me any more references. 
Thank you!
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I haven't worked with this particular cell line before but I have worked with other cell lines from ABM in the past. I did not encounter any problems with them.
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We have homogenized Taenia crassiceps larvae and are puffing the homogenate onto pyramidal neurons during whole-cell patch-clamp recordings in organotypic hippocampal slice cultures. We need to put the puffer pipette very close to the neurons - ie the neurons move during the puffing - we see obvious depolarization ie 10 - 20 mV worth, that is not blocked by glutamate receptor blockers (kynurenic acid, AP5, CNQX). The pH is roughly between 7 and 8. Osmolarity of the homogenate is 300 ish. Also the K+ concentration within the homogenate is 4 mM and the effect is there when using a caesium internal. Is what we are seeing  an artefact?  What substances cause neurons to depolarize, what should we be thinking of as causative agents that might be in the homogenate?
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Most likely the homogenate contains glutamate as it is near millimolar concentrations in cytoplasm which will activate glutamatergic receptors in the pyramidal neurons. Glycine concentration is also likely sufficient to activate GluN1 subunits of heteromeric NMDA receptors. I suggest coapplication of ionotropic glutamate receptor antagonists, eg. CNQX and APV.
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Dear all,
I am recording sEPSC in layer V neurons in mouse mPFC, but I found some abnormal responses showed as attached pics. They look like epileptic discharge in presynaptic neurons,  is there any e-phys expertise can tell me what was wrong with my recording?
I held the neurons at -70mV, and add picrotoxin 100 uM to the ACSF.
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Hi, what do these look like under CC? I suspect they are the compound EPSCs that cause paroxysmal de polarization shifts. I think this is a result of ictal network activity you're getting from applying picrotoxin to the entire slice.
If you're using it to disinhibit everything on purpose, you are in a tricky place... 
If on the other hand you just use the picrotoxin to block sIPSCs, I'd recommend local application (ytube) to avoid the paroxysmal activity that's likely causing these giant currents! 
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I am looking for a robust marker for the gut neurons in Drosophila. I found that both 22C10 and Tuj1 can be used. Is there maybe another one that someone could recommend?
Thank you very much!
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Dear Anderson,
I have not tried this on so for. Thank you for the hint!
Annekatrin
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I have routinely carried out calcium imaging experiments in cortical neurons 9-11 DIV for 24 h with no problem. However I am now culturing mature hippocampal neurons (DIV 14-16) and the cells seem to deteriorate within 1-2 h in this buffer. Is there any special buffers which should be used for imaging of mature hippocampal neurons?
The buffer I use (& remade just in case) is (in mM):120 NaCl, 3.5 KCl, 0.4 KH2PO4, 20 HEPES, 5 NaHCO3, 1.2 Na2SO4, 1.2 CaCl2 and 15 glucose, pH 7.4.
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The solution suggested by Karthik works well. If you wish to perform the imaging for 24 hours or so then it is better to bubble the solution and also maintain proper level of humidity;  If you are using Leica microscopes then they have a special chamber to establish required humidity level. In my experience Hippocampal Neurons appears more robust than the cortical neurons and so you should be able to perform the experiments  easily!  
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at the moment im working with cortical neurons from rat embryos. 3 DIV neurons looks like the photo i atached. i wanna know how to improve disociation, im using trypsin 10x. but the majority of plates looks conglomerate.
i aprecciate any advice you have
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Dear Daniela,
Thanks for your reply.  The reason I asked is that some of the axons are growing in a very fasiculated manner, and that sometimes means that they are not happy with the substrate.  In a similar manner, if the neurons are not happy with the substrate, they tend to clump more.  However, in my experience, cortical neurons are usually pretty happy with poly-D-lysine, unless the glass has not been well cleaned before coating or the PDL has gone bad.
You don't mention what technique you are using to dissociate your cells, or for coating your glass.  You might be interested the answer I posted to this RG thread, as well as some of the other answers:
Good Luck!
Jill
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Dear Researchers,
We are working on acute hippocampal slice preparations for electrophysiological studies using MEAs' (P28 Wistar rats).
We are getting good viable slices with spontaneous and evoked signals.
We wish to know various drug testing protocols using acute hippocampal slice preparations for electrophysiological studies in view of increase in spike rate, amplitude, burst analysis, LTP, LTD etc.
Would you please share your expertise with any standardised drug testing protocols using MEAs'
Thanking you,
Best Regards,
Dr. Grandhi V Ramalingayya
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I guess you only have the option of bath perfusing a drug and washing it of after sometime.
However, you would have some circuit level artifacts ie due to the activity on certain neurons which might be connecting to and hence activating some other neurons on the dish.
But to start with its not a bad idea.
Regards,
Debanjan
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I am doing currently intracellular recording in microdissected islets of Langerhans. It would be interesting to me to consider a collaboration.
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Hello Juan,
Thanks a lot for noticing in our work. Your project is potentially interesting for later stages of our work, but not any the moment. 
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Currently trying to slice mice brain from 200um slices into 40um slices for IHC. At times it works perfectly fine, but other times there are complications where the slices do not stay intact, or the whole 200um brain slice falls off. We use 30% sucrose and dry ice as a glue to the machine. I would like to ask if anyone has some tips and tricks to improve the quality of the slices. 
The 200um slices were introduced to dopamine and then left in PFA. In case of not proceeding to slicing immediately, we store them in Anti-freeze and then wash it with pbs thoroughly prior to slicing.
- Any environmental factors that may have a big affect on the slicing (perhaps temperature wise)?
-Any blades that are optimal?
-The timing of PFA incubation?
Any tips would be very appreciated!!
Thanks,
Lynn
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How long do you fixed in PFA? or did you perfused the animal? I have not used the anti freeze, but I can reccomend the last wash be with 30% surcose before embede in OCT medium to slice.... I have did it already and it works.... you have to make sure to extend your 200um slide in an uniform position before freezze... in order to obtain your 40um slices. Good luck!! 
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Hi collegues,
I'm trying to study the glutamate release probability in hippocampal cultured neurons. I started using hyperosmotic extracellular solution (1M sucrose) but this method evaluates the RRP inducing Calcium-independent release of glutamate. Do you know any methods to study Calcium-dependent release?
Thanks in advance for helping me
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Hi M. Pilar,
i'm searching for an electrophysiological method to study glutamate release. Thanks for your input anyway.
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Dear all,
I'm looking for a reference which tells me when NMDA receptors are functionally developed in murine cortical neurons in vitro. I could find many article about hippocampal neurons and also about rat neurons. However, so far I didn't find a reference stating at which DIV NMDA (and AMPA) receptors are functionally developed in cortical neurons. I'd be glad if anyone could name a reference!
Thx!
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Hi Anne-Sophie,
Maybe not the most complete study, but perhaps a good place to start:
Best,
Andrew
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Does Cas9 cleave ssDNA?
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SpCas9 could cleave complementary ssDNA in vitro. You could found the result in Fig.1B of this paper, which is named "A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity".
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We recently had a discussion if cell culturing lead to a higher basal activity compared to in vivo neurons which might occlude an effect in increased higher amplitude of the mEPSCs. You guys ever heard about this theory? It sounds reasonable to me but I can't find basic literature about it.
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Even more confusing is that during cell culture conditions, is it possible to have "autaptic" connection and to generate autaptic currents under "whole-cell" current recordings? I think that more importantly, because neuronal cell is rather small, how to determine cell-attached or whole-cell recordings is potentially important.
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Is my first time working with stem cells and I don't what kind of stem cells are better for this. 
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It would be helpful if you specify which kind of neurones do you want to obtain. There are plenty of protocols published and some commercial kits that can be useful (I would recommend you to better find a protocol than a commercial kit since they are quite expensive). 
And also what kind of stem cells: if you want to start with pluripotent stem cells (embryonic or iPS) or if you're starting directly with neural stem cells, if they are human or from any other source, etc. Then I guess people might give you more specific answer.
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Neuronal cell lines
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This is a difficult and complicated scenario. However, you could simplify the situation by deciding upon the research question you are trying to answer. You then employ the appropriate tools to answer your research question or to test your research hypothesis.
First, note that there is a difference between a neurotoxin and a neurotoxicant. A neurotoxin is a substance produced by a living organism, such as tetrodotoxin produced by the puffer fish. A neurotoxicant is a synthetic substance such as the nerve agent sarin.
You need to decide what type of acute neurotoxicity you are interested in producing and whether or not the toxicity is reversible.
Finally, note that a neuronal cell line is not really neuronal, given that cell lines consist of transformed cells that can divide. Neurons (except for neuronal stem cells) are post-mitotic cells. In addition, neurotoxicity seldom is expressed within individual cells; it depends upon interactions among and between cells.
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I see the AMPAR surface level increased by Foskolin and Rolipram goes back to the level before the chemical induction in the cultured hippocampal neurons. Is it supposed not to happen or else?
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Hello,
I understand how does Forscoline increase the superficial AMPAr level but I don't see how can it contribute to  the receptor stabilization on the membrane because the Ca entry trough synaptic receptors (mainly NMDA) is required. Therefore the AMPAr internalization is supposed to happen after chemical treatment.
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Any good paper/publication regarding this would be helpful.
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I am trying to see, in a culture of hippocampal neurons, cells that are metabolically active from those that are not. I could do it by confocal microscopy, but I think the cytometer will give a much more rigorous and rapid account. Thank you very much if you have an answer.
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Hi Pablo, "metabolically active" as in "alive" ? The classic approach is an acetomethyl (AM) version of a fluorescent dye becoming trapped inside cells alive enough to have functioning esterase. To dissociate neurons from a monolayer culture, I would suggest something gentle like dispase or accutase. We are also working on a similar approach so I will keep you posted once we get a stable protocol. I hope this helps.  
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Background: I am interested in studying the electrophysiological properties of PV interneurons in brain slices of adult mice. I bought a PV-eGFP line (CB6-Tg(Gad1-EGFP)G42Zjh/J) from Jackson to perform this experiment and patiently aged the mice. Unfortunately, there seems to be some epigenetic silencing of the eGFP with age (I blame Jackson for not properly documenting this, even though apparently many people have complained about this. Be wary of this line)! So I have all of these aged transgenic mice, but almost fluorescence anywhere!
Question:
Instead of wasting my efforts and sacrificing these aged mice, I would like to see if blind patching may be a viable alternative. Do any of you guys have any advice on how to identify PV interneurons using strictly DIC? We will ultimately be validating the identity with current injections to see spiking patterns, but I want to increase our chances of getting the right cells with DIC. I was told by some that PV interneurons tend to have smaller and rounder somas. Can anyone validate this? Or direct me to papers where they do blind patching on PV interneurons?
Thanks!
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Hi Ruoqi,
The answer is yes. It is definitely doable to patch PV+ Fast-spiking interneurons(FSIN) without fluorescence.But the difficulty is depend on the brain region. In dentate gyrus, you can Identify FSIN by there soma size, which is much larger than granule cell. However, in CA1 region, the FSIN soma size and shape are very similar to pyramidal cell, so it is much harder to find them with DIC. You should find out whether FSIN have special shape/size/location in your target region.
About the age, PV is not expressed in certain age. You can try to use lhx6 or GAD as promoter to drive GFP. So you can identify interneuron first, and further identify PV neurons with their intrinsic electrophysiological properties.
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I need to mix human neurons with mouse neurons or astrocytes. So I have to pass and count human neurons, but I can immage that is very dangerous and they tend to die. Let me know if somebody has set up a protocol. Thanks
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Neurons are quite robust and can be replated by trypsinization and centrifugation just as you would passage any other cells. After adding trypsin you have to monitor every 3 minutes that the cells and neurites are detached before removing for centrifugation.  Not more than 6 -8 minutes max.  Neurons being postmitotic terminally differentiated calls will not divide, but they do regenerate.
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Looking for any normal brain cell line.
Can collect if in west mids or pay for postage if further.
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I'm afraid there is no such thing as a "normal brain cell line". I think you'll have to be a little more specific about what you're looking for....
best,
Agnete
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Hi all, 
I just moved to a new lab and started cell cultures again. We are culturing hippocampal neurons from E18 rat embryos. The cells seem to mature at normal rate and I can see synaptic activity already at DIV 11 (yay!). However, I saw a lot of round circles on the coverslips. Attached is a picture of the neurons at DIV 7. Do any of you know what could be the origin of these things? I think they are most likely dead cells since the size is about that of neurons, and I can also saw some similar texture that have the shape of a soma. But it is really weird since they do not disappear over time even after we change media, they do not go away. Do you think they are harmful for the other alive neurons? In the past, I saw these when we do Banker style and I thought the astrocytes layer somehow prevent liquid flow in between but this time we only have neurons. How can we get rid of them? Any experience or advice would be greatly appreciated!!! :) Thank you all! 
Sincerely,
Huong
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Hi Angelica, I haven't completely solved the issues but found a few factors that affected the appearance of these round circles. Just to clarify, we are doing Banker style culture, with an astrocyte feeder layer in the bottom and neurons plated on glass coverslips facing the astrocytes. When the astrocytes are too fresh, or when we had bad B27 lot numbers that promote growth of non-neuronal cells, there were way more dead neurons/circles thingy. We had tried to lower the density of the astrocytes feeder layer, and also tried different B27 lots to minimize the issue. Another factor that contributed is the distance between the astrocyte layer and the coverslip with neurons. I learnt that if there was not enough distance between the two, there were more dead cells. So, I just simply made bigger wax feet to increase the distance. Hope this helps!
Huong
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I'm just learning about current tracings and that it's important to compensate for pipette capacitance when trying to get a better picture of the current-does this have any effect on the reliability of a mIPSC trace? I can't see much of a difference in the actual recording when switching back and forth. 
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Hello Taylor,
Well, let me ask you some questions. I'm not sure that we mean exactly the same things.
1/ You're talking about pipette capacity compensation or t RC compensation of the cell ?
If so,  compensating for it should increase mIPSC kinetics and amplitude but not that much unless actual kinetics are fast and compensation is ideal. The problem is that it is always impossible to fully compensate capacitance when working on submerged slices because the (fast) compensation range is limited (usually 1-10 pF, art least on the axopatch 200B). When recording on slices under the microscope objective, the electrode tip and shank are fully submerged by the perfusion saline which would give a large pipette capacitance. Remember that pipette capacitance would correspond to the submerged surface of your electrode. Because under the objective the liquid meniscus could be large, the submerged part of your electrode would be large as well. 
2/Do you succeed in compensating until the recordings start to show oscillations ?
3/Do you paint or coat electrode with sylgard or bee wax or dental wax (that's very helpful but painful if you use sylgard ) ?
Of course if the actual kinetics are relatively slow (do you work at RT ? ) , you may not see an improvement but the rising time of mIPSCS is usually fast (< 1ms?).
Regards
Fabien
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I am specifically looking to sort for microglial cells from brain. Any comments/technical suggestions will be helpful. 
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Thank you both for your responses. I had not heard of sorting in frozen tissues and was curious if anyone has tried it before. Thanks for addressing my question and the alternative suggestions. 
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Is it possible to chemically link a small peptide to the side chain of a Heparin sulfate proteoglycan? I know there are antibodies available, but I am specifically looking for a small peptide with less than 10 amino acids.
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It probably is possible, but good luck documenting it. The Heparin sulfate molecule is an extended mess of ill-defined and ill-ordered sulfo-glycans strung on a protein core. Its best use is as a bulk molecule for taking up space. Son't waste your time getting into its fine structure - there really isn't any. Just attempt you linkage, and then see if the product has immune properties that are helpful to you.
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I have this question just out of curiosity. I read a couple of publications in which a saporin-conjugate is used to kill a specific neuron population, for example GABAergic neurons by stereotaxic injection of antiGAT-1-sap into the brain region of interest.
I guess the specificity of saporin-conjugates mostly are determined by specific receptor-ligand binding. This also means saporin can enter the cell or subcellular structure where this binding present. In the example mentioned above, is it also possible that antiGAT-1-sap kills the passing fibers/axons of long-range projection neurons that also GAT1 positive?
Let me know what you think, especially for those have employed a saporin-based methods for brain lesions. Thanks a lot! :)
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Dear Vladimir,
It is a stimulating article, thank you. "Unmodified saporin cannot enter cells but at high, micromolar concentrations it can trigger apoptosis due to background endocytosis."
Considering the very active vesicle recycling at synapses, the axonal protein synthesis and axoplasmic transport, it's then quite likely saporin can enter the axon, even being transported back to distal cell body.
It is better if we can have some input from who actually make lesion of neurons by toxins (i.e.Tetanus toxin, botulinum toxin). Probably, the concentration of toxin, the survival time  are among the critical factors for a local lesion.
Best,
Jiahao
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I have brain frozen mouse brain areas (frontal cortex, hippocampus and hypothalamus) and I would like to extract RNA from them using the Trizol or the RNeasy kit. Is it sufficient to use a insulin syringe and needle to homogenize these brain pieces (mm for less than 20 mg tissue) or is it better to use a tissue lyser? What approach will lead to RNA samples with the highest RIN?
Thanks
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Syringe and needle is sufficient, and the advantage is that you are only using disposable material, avoiding possible cross-contaminations. Be careful with frozen tissues: RNase will start acting as soon as they melt. So I prefer to disrupt fresh tissues, and to freeze the homogenized lysate (high salt buffer inhibits RNase) if I cannot do the extraction the same day.
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How can I measure Emax percentage in a dose response curve when dealing with EC50 or IC50 values of my agonists from GraphPad prism data/ graphs?
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In your 'results' sheet you get bottom and top values (lines 3 and 4) representing the upper and lower asymptotes of your DRC.  Set the difference to 100 for your reference curve/compound and relate values from other curves to it.
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I am looking for a staining method to label dead neurons in the ischemic mouse brain.I use coronar cryosection from mice which were subjected to transient MCAo and reperfusion. I tried NeuN+TUNEL but unfortunately the dead neurons in the ischemic hemisphere lose too much NeuN.
Do you have any suggestions or even a protocol?
Thanks in advance!
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If you are looking for focal ischemic changes TTC staining gives you clear idea of infarct area but for global ischemic changes TUNEL/FluoroJade/H&E/etc will be helpful. 
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Hello, I want to detect by ELISA the presence of an administrated anti-Abeta antibody in mice brains. The mice were treated with a recombinant humanized antibody intraperitoneally and I want to know if (and how much) antibody would have crossed the BBB.
My doubts concern about the most appropriate lysis protocol of the brains for detecting those antibodies by ELISA.
If there is a more adequate way to perform the experience, I am open to advises and comments.
Thank you very much!
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I did not use TX100 and protein column G (for low concentration it is a good idea), however I did the same to check the rate of protein penetration through the BBB. Good luck!
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I am just wondering the identity of these cells. These look like neurites. These are GFAP and S100 positive and also  show immunopositivity towards class III B tubulin.
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Hi Varun,
the only thing I may tell is that at some time points in differentiation astrocytes (precursor cells) express GFAP and S100 and so do Schwan cells. For example, in some of my cell cultures embryonic astrocytes also express MAP2 subunit c and are labelled by anti-MAP2 (neuron marker). 
May be the link below will help you.
Best wishes,
Christina
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In case of adverse effects of  lithium carbonate
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Dear Slim,
Yes indeed. lithium orotate has a mood stabilizing properties. The following text describes some of irs properties and advantages over the traditional lithium salt (lithium carbonate).
Medical doctors have found servings of lithium orotate 80-90% lower than the orthodox Pharma-lithium serving for major depressive disorder, also known as clinical depression, unipolar depression, major depression, or unipolar disorder. 50-70% of patients have shown fair to good response with lithium use for depression.
According to Jonathan Wright, M.D., America’s top authority on lithium orotate, a total daily intake of 30 milligrams of elemental lithium will have unnoticeable effects on serum lithium levels, with levels usually residing in a non-detectable range. Even 40 mg per day appears to be completely safe, presenting no negative side effects or signs of toxicity.
Administering lithium orotate up to 40 mg per day to be completely safe (without negative side effects or toxicity) and absolutely effective in the control of numerous mental, neurological, and physical conditions. Lithium orotate, is preferred because the orotate ion crosses the blood-brain barrier more easily than the carbonate ion of the pharmaceutical lithium carbonate. Therefore, lithium orotate can be used in much lower servings (e.g. 5 mg) with remarkable results and no side effects. 
“The lithium salt of orotic acid improves the effects of lithium several-fold by increasing lithium bio-utilization.” -Ward Dean, M.D., author of “The Unique Safe Mineral with Multiple Uses”
 Lithium orotate has also been successfully used in alleviating discomfort from migraine and cluster headaches, improving low white blood cell counts, juvenile convulsive disease, alcoholism, and liver disorders. Lithium Orotate is extremely safe, with no known adverse side effects or drug interactions.”
“Prescription lithium is poorly absorbed by the cells, where it needs to be to do its job… Because it is so poorly absorbed, blood levels need to be fairly high to “drive” it into the cells. Unfortunately, these “therapeutic” blood levels are dangerously close to the toxic level. That’s why patients on prescription lithium need to be carefully monitored… Successful serving with lithium orotate is measured by clinical effects on the patient, rather than by blood levels.” -Ward Dean, M.D.
“Lithium Orotate will not cause weight gain, nor will it cause sedation or sleepiness.” -Ward Dean, M.D.
Lithium orotate therapy was seen as relatively safe, with minor adverse side effects seen in some patients (muscle weakness, appetite loss, mild apathy). For these patients, symptoms subsided following the reduction of lithium orotate administration.
-A study titled: “Lithium orotate in the treatment of alcoholism and related conditions.” Available without a prescription, lithium orotate has been marketed as an alternative to the prescription known as lithium carbonate. In lithium orotate, lithium is joined with an orotate ion, rather than to a carbonate ion.
“Lithium orotate is a highly bioavailable form of lithium that is available as an over-the-counter dietary supplement.” -Linda Fugate, Ph.D., author of “Lithium’s Potential Role in Preventing Alzheimer’s Disease”
“In small servings (15 mg/day), lithium orotate has been shown to protect the central nervous system.” -Dietrich K. Klinghardt, M.D, Ph.D.
For more details on this agent, please use the following links:
Hoping this will be helpful,
Rafik
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To what extend the white spot can affect the vision of mouse and do you have better practice to minimize the occurrence of them?
P.S: I keep the eye covered with animal eye gel, try to minimize the direct light from the lamp, but sometimes the white spot becomes so obvious within 1hr that it almost occupy most of the eye.
Many Thanks!
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Hi Janet, the white spot usually disappears gradually during the recovery stage. I later find a complete wetting of the cornea during the whole surgery dramatically diminishes the chance of having white spots. In addition, it is good to avoid a direct surgical lighting towards the eyes.
Thanks for putting forward a possible cause by the hypotension. The cardiac output diminishes for sure when the mouse is anesthetized. 
Good luck to your research!
Jiahao
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I'm trying to analyse NMDA-currents recording at -70 mV from dissociated-cell cultures of hippocampal neurons from embryonic rats (14-15 DIV) with a density of 40000 per cm² (I've been trying also with 90000 per cm²). Currently, I've been using this external solution: NaCl 150 mM, KCl 4 mM, HEPES 10 mM, Gluose 10mM, CaCl2 2 mM, MgCl2 0 mM, pH: 7,3, osmolarity around 300±5; and internal solution: CsMeSO3 107 mM, CsCl 10 mM, NaCl 3,7 mM, TEA-Cl 5 mM, HEPES 20 mM, EGTA 0,2 mM, ATP-Mg salt 4 mM, GTP-Na salt 0,3 mM, pH: 7,3, osmolarity 298-300. Last time that I managed to achive NMDA-currents peaks (average of the peaks aplitude=25 pA), after having reached the gigaseal with pipette resistance between 3-4 MOhm, the cell had these parameters: Cm: 42,37 pF, Rm: 340,3 MOh, Ra: 15,9 MOhm, Tau: 641,9 micros, Hold: -143,8 pA. I use Clampex software.
Many thanks.
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Hi Pasqualino,
Just want to remind you that CNQX is not perfect in isolating NMDA currents, because other than blocking AMPA/KA receptors it may also reduce NMDA open probability. NBQX would be a more clean AMPA/KA antagonist. I recently encountered a similar problem, and i found this explanation in some papers.
Bo
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Hi
I am facing difficulties in genotyping mice carrying genotyping alleles. Can someone provide me with an established protocol and primer pair for neomycine genotyping? Thanks heaps in advance
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Thanks 
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To extract the primary cells in mouse brain.
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Sorry Guosheng, what's a complicated cell in this case?
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Hey everyone,
I know the question seems paradoxical, but I am currently trying to measure sEPSC in the medial Hippocampus CA1 cells in adult mice voltage clamped at -70mV, and I find that there are these huge (1500~4000 pA) currents that pop up every minute or so (image attached). The internal solution is CsMeSO4 based and contains 5mM QX-314 (alomone labs). From my understanding, the voltage clamp and QX-314 should both stop the clamped cell from firing, but it does. Frustrated that I was, using the same int sol I changed to current clamp and found that there are depolarizations that go up to 10mV, which is why I am saying that the cells are "firing." Has anyone experienced this? As a last resort I borrowed some internal solution with the same composition from another lab, but got the same results. The alomone lab website, by the way, says that the particular chemical, at 5mM the Na current is reduced by ~20%, and complete blockage of sodium currents require 50+mM concentrations. But from what I know the conventional knowledge is that 5mM is enough to block them, so I am at a complete loss as to what to do. Any help would be appreciated. Thank you all in advance!
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Dear Haram,
The neurons you are trying clamp are very large and therefore there is a problem with space-clamp issue, meaning that you are not actually fully clamping the cell at the holding voltage outside the immediate location of the electrode tip. Action potentials are coming from the axon hilux are sometimes difficult to clamp, especially if you are using small tip (high resistance) glass micropipettes. What is your access resistance readings? My recommendation would be to use larger tip electrodes 2-3 megaohm (~1 micron) in the bath solution. The larger the electrode-membrane interface the lower the access resistance will be, meaning you will be able pass larger amounts of current that will give you better voltage-clamp. The access resistance ideally should be less than 10 megaohm, especially in neurons with larger dendritic trees. This should also help passage of QX-314 to the intracellular compartments easier. For inspiration see my following publications.
Best wishes, Refik
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Hi, I am looking for a tag/fusion protein for myelin proteins MBP, PLP and MOBP that does not interfere with their physiological function (or their CNS/cellular localization). I considered an HA-Tag (Aggarwal et al., 2013), V5-tag or Myc-tag rather than a GFP-fusion protein. The idea is to identify the protein of interest biochemically and by immunocytochemistry/immunohistochemistry in the presence of endogeneous myelin protein (potentially from a different species). Very much appreciate any suggestions!
Best regards,
Jens
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Thanks Sebastian. The first step would be to transfect primary CNS cultures with focus on oligodendrocytes. I thought about an HA-tag and an AAV8-based viral carrier.  Detection methods would be ICC, IP's and WB. Any advantages of myc vs. HA?
Thanks again,
Jens
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I would like to be able to count cells in the rat and mouse hippocampus (for example)... most of our slices are ~10-50 um thick, and it would be nice to be able to use something fluorescent (e.g., DAPI), but if that's too cost prohibitive, it's not necessary
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Hello Rico.
It is not so difficult to count cells in tissue slices.
You could double-label the cells of interest with a specific cellular marker and DAPI. What you definitely need is a fluorescence microscope. You are working at a large university. I can hardly imagine that there is no scientific group with such a microscope. There may be even a core imaging facility available (f.e. the Bakewell NeuroImaging Laboratory). You have to broaden your network at your university and contact those groups. It is worth asking, they wan´t cut your head off ;-). Microscopes are very expensive.
If you have taken the images - I think 20x magnification would be sufficient, you can use several methods for counting nuclei via ImageJ (a java-based free software):
- manually: with the CellCounter PlugIn
- semiautomatically: with the ParticleCounting PlugIn
Here are some links:
- Blakewell NeuroImaging Laboratory: http://thalamus.wustl.edu/Facilities/Bakewell
- ImageJ download with a lot of PlugIns uncluded: http://www.uhnresearch.ca/facilities/wcif/imagej/installing_imagej.htm
Hope this helps!
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The thing is, I need to sample the different layers of a sheep cortex at different fetal stages, but I need to do it in an unbiased way, for I have to correlate gene expression from one point of it with the gene expression of the exact same point but at a different fetal age.
Any paper you could suggest for a starting point?
And no, I'm not planning to do an immune approach or in situ hybridization, because for that it'd be easier to use a complete slide. I need to extract many samples from that brain (and some other fetal stages as well), extract the RNA and run a microarray. 
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Benjamin you rock dude =) This is just what I needed. I'll try to pull it on fetal sheep somehow.
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Stereotactic Intracranial injections are extremely time consuming (Can only do 10-12 animals in a full day), are expensive- requires purchase of drill, dissecting scope, stereotactic apparatus, automatic fluid injector. Since many papers use different brain locations for injection of tumour cells and there is still significant error due to brain curvature etc., the exact location of the injections may not be crucial.
Meanwhile, guide screw injections are considerably faster and cheaper- only requires the drill, the accessory screw pieces and perhaps a dissecting microscope. It would also be much more practical for the sake of performing injections in large cohorts. This technique has been used in many high impact publications, particularly Zhang, 2015 in Nature (Exosomes PTEN Brain metastasis paper).
The flaw with this technique are that they are less precise than stereotactic injections, and this may lead to larger variation in tumour growth between animals. However, for the above stated reasons, I am wondering if it is worth the gamble.
Does anyone have experience with the guide screw technique, or both techniques that can provide input?
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Hello,
I do not have experience with work with tumor cells. However, I have almost 30 years of experience doing rodent surgeries. With respect to recovery of the rats or mice, the neurochemical environment, neuronal cell types, and glial cell types, there is a large degree of variation from one brain region to the next. For example some regions of the brain have proliferative zones and others do not. If one were to hope for refined answers to their questions, it seems like a very good idea to track the precise location/region into which you injected each time. There will be far more precision using stereotaxic injections. The Nature paper you mentioned - spoke globally of brain and other organs (the CNS is very diverse relative to other organs - so treating it with more precision is the norm and will likely yield better results).
So, it really depends on how precise an answer you need and how reliable you want your data to be.
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We want to observe by fluorescence microscopy or light microscopy the parallell fibers in rat cerebellum. Is it better to use sagital or coronal sections? Any suggestion for a marker?
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Dear Natacha,
If you want to see a long stretch of PF (intervaricosity distance study ?) a coronal or transversal section seems more appropriate than parasagital. As for marker, you probably do not want to label ALL the PFs, it would become too messy to see anything. I would suggest to place a very small DiI crystal on the GL or inject the GL of your fixed slice with a bolus of Fast-red (slightly less lipophilic than DiI). If the crystal/bolus is small enough and you if you leave your slices rest at 10C long enough (w/ azide?) you should get a subset of brightly stained PF. I hope this helps. Good luck    
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ATP released from astrocytes is degraded to adenosine and activates presynaptic adenosine A2 or A1 receptors that leads to an increase or decrease in its release probability (Panatier et al. , 2011). Now the problem is:
After secretion of ATP by astrocyte:
Which mechanism is activated A2A receptor on presynaptic neuron?
Which mechanism is activated A1 receptor on presynaptic neuron?
Which mechanism determines that what kind of adenosine receptors on the presynaptic neuron (A2A , A1) should be activated in response to astrocyte adenosine secretion?
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You should send your question to Prof. Harald Sontheimer who is registered on RG.
Best regards
Robert
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I plated a 96 wells plate with SK-N-AS cells, which I then treated to induce a lysosomal storage disease. Then, I treated the cells (in threefold) with different drugs that would hypothetically lower cholesterol levels. After, I measured cholesterol levels and, because the cell growth seemed to be influenced by certain drugs, protein levels of the cells.
How do I use the protein assay results to cancel out any differences in cell confluencies?
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I already measured the protein in micrograms/ml. I divided [cholesterol] by [protein]. Now all cholesterol seems about the same (also, none of the results are normally distributed). I guess I should treat the cells with a higher concentrated solution.
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I am culturing Schwann cells and need to analyze the conditioned media
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What's kind of secretory protein you plan to normalization?  I think that you need to set up the positive and negative controls, as well as internal control at the first step.  Second, you should know how much the concentration of this secretory protein can be expressed, then you will using more high sensitive of monoclonal antibody for this secretory protein measurement. Any standard proteins can be used for your secretory protein normalization.  Let me know if you find any new standard proteins?  Thanks!
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Can anybody share the protocol for isolating microglia from mice brain as I need to sort the isolated microglia for RNA-Seq purpose.
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I have successfully isolated microglia from adult rat brains using: Frank et al. 2006 (Journal of Neuroscience Methods 151: 121–130).    It is much faster than other methods that I have used.
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I want to compile a list of acute pathological or physiological events that last no more than 5 min. Currently, I can only think of cortical spreading depression (CSD) and seizure that qualify for this standard (very short, duration less than 5 min). Status epilepticus maybe OK, but I still think it lasts too long. Ischemia is not qualified for the same reason.
Can some one give me more examples? Any physiological and pathological events will be fine, as long as they occur acutely and are short-lived.
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Do you go for pathophysiologically or clinically defined events? I didn't get it quite clear when reading your question.
As a clinician, I would add hemifacial spasm and trigeminal neuralgia. Status epilepticus is defined by either 5 or 30 minutes ongoing seizures activity, so it won't qualify. Neither does spreading depression, at least when you translate it into complicated migraine, where the corresponding aura can last a lot longer. Ischemia is qualified if you consider TIA or syncope, which is finally transient global cerebral ischemia.
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The blocking reagent from Roche, which used to be from Boehringer Manheim and now is being held hostage by Sigma, has been backordered since August 2015. I am doing whole mount in situ hybridization on Xenopus laevis embryos. Does anyone know of a good substitute? I tried 5% milk and I got a lot of background staining. We are running out of our backup stock of 10X blocking reagent fast. Please help!
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Thank you so much! I will try that combination and see if it works.
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Do MOIs as low as 0.5 work well, or are MOIs of 4 to 6 needed?
Could anyone help with a standardized protocol. Many thanks!
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Lentivirus should infect neurons quite efficiently, but I doubt an MOI of 0.5 will get the job done. If you already have virus to hand, just try MOIs ranging from 1-