Cellular Neuroscience - Science topic

Hi everyone,

I’ve been trying to get my review article published for over a year now, but I keep running into roadblocks. I don’t have support for open-access fees, and I’ve submitted it to more than 10 subscription-based journals, but they’ve all rejected it, saying it’s not suitable for publication.

I’m looking for someone who could help me get this article published. I’m not concerned with the impact factor, and I’m fine with any authorship arrangement—you can be the corresponding author or co-author if you want. I just really need help to get it out there.

If anyone can offer advice or help, I’d really appreciate it!

Thanks a lot

Irum

PhD student (Cellular Neuroscience)

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!

Dear all, I am trying to do a synaptosomes preparation from Postmortem Human Prefrontal Cortex (frontal). I read few articles, they used 1 to 5 gram at the beginning. I started with low amount of frozen brain, around 300 mg (PMI < 4 hours). From 300 mg weight of prefrontal cortex, I generally obtain ~25 micrograms of total protein from isolated synaptosomes. 

Is anyone have any idea to increase the yield at the end (beside increasing the weight of the brain)? I do not have a high amount (weight) of human brain.

Each minced prep is immediately homogenized by applying 20 slow stokes using a teflon-glas tissue grinder (grinding chamber clearance is 0.15 mm). Then I use layering of discontinuous sucrose gradient.

I am thinking to use a glas-glas tissue grinder (grinding chamber clearance is 0.025 - 0.076 mm).

I welcome any idea.

Cheers,

Stella

Suppose we want to search an object within a visual field in which the desired object is not essentially existed in it. What would be the activation of the FEFm neurons?

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. 

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

I’ve been having numerous issues with achieving stable baselines recording from the TA-CA1 synapse from juvenile (P12-P24) rat hippocampus slices. In addition, when applying drugs such as antagonists/inhibitors which should not show any effect on baseline, I have been seeing gradual increases in synaptic transmission that differ from what other students have previously shown in my lab.

I cull my rats by cervical dislocation and slice in ice cold sucrose aCSF and allow the slices to rest for 1 h at RT in regular aCSF. I then stimulate and record from the TA-CA1 and my first slice usually takes 2-3 hours to stabilise. I oxygenate my aCSF for at least 40 minutes prior to putting a slice on the rig and I use a platinum harp to hold it down in the bath. My rig uses a gravity feed system and the flow rate is 2.5 mL/min. My recording electrode is filled with aCSF and I bleach the silver wire every few days.

When the slice eventually stabilises for 20 min, I add my drug which has been oxygenating for at least 10 min. I can often see strange increases caused by the drugs that have not previously been seen. I thought it might be down to changes in oxygenation but I’ve been keeping all of my solutions in similar sized cylinders and have increased my oxygen so that everything is saturated.

Can anyone advise me how I can improve this and shed some light onto why I am seeing such instability and increases when switching drug?

Any help would be much appreciated, as I feel as though I’ve exhausted all ideas at this point.

Thank you!

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.

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

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?

I'm looking to collaborate.

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

If so, has the use of the GloChicks been more effective than using a non-transgenic chicken, in terms of imaging quality?

Thank you.

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.

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?

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!

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!

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:

PI:  Laser 561 nm; Filter 670/30

AnnexinV: Laser 488nm; Filter 530/30

Controls:

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)

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?

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

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!

I am studying the establishment of synapsis in N2a cells, comparing synapsis protein expression in differentiated and non-differentiated N2a cells.

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.

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

Cells not easy to transfect

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?

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

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!

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?

I am thinking on using NeuN? Any comments, suggestions?

Is there something specific about neuronal membrane composition at the molecular level that makes it different from other cells membrane?

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.

 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!

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?

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.

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!

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.

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

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,

I am doing currently intracellular recording in microdissected islets of Langerhans. It would be interesting to me to consider a collaboration.

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

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!

Does Cas9 cleave ssDNA？

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.

Is my first time working with stem cells and I don't what kind of stem cells are better for this. 

Neuronal cell lines

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?

Any good paper/publication regarding this would be helpful.

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.

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!

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!

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

Looking for any normal brain cell line.

Can collect if in west mids or pay for postage if further.

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

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. 

I am specifically looking to sort for microglial cells from brain. Any comments/technical suggestions will be helpful. 

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.

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! :)

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

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?

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!

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.

In case of adverse effects of  lithium carbonate

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!

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.

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

To extract the primary cells in mouse brain.

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!

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

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

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. 

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?

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?

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?

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?

I am culturing Schwann cells and need to analyze the conditioned media