Questions related to Molecular Neurobiology
I am interested in Neurodegenerative diseases, particularly Dementia, and I would like to study the disease basic neurobiology. This will be a helping hand for me to study Dementia well.
Recommendations could be Books, Articles, Lectures, Animated videos..etc
Thanks in advance!
When I search for the dilution ratios of a number of primary antibodies for IHC (immunohistochemistry) studies, I usually see that the ratios are somewhere between 1/50 and 1/500. Apparently, this range (1/50 - 1/500) generally fits for IHC studies. In case of those antibodies, when it comes to applications for other methods such as western blot, immunofluorescence, the more diluted stock solutions still seem to work (somewhere between 1/500 - 1/1000). Is this just a coincidence, or is there really a linear relationship between application method and dilution ratios ?
If so, for a IHC study, I would like to know if it's a good idea to prepare several dilutions (more diluted) different than the manufacturer's suggested dilution. If the solutions diluted more than the recommended ratio still work just fine, then it would be better to use this way, because by doing so, much less compound would be wasted.
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?
I'm trying to get subcellular fractions (specifically, I only need the nuclear fraction) from neuronal tissue. However, all my current samples were flash frozen and stored in -80C subsequently (for whole cell lysate, which was my original intent for the samples).
Does subcellular fractionation for nuclear protein require fresh tissues and cells? Will flash freezing might cause ice crystals to form and pierce the nuclear membrane, so I wouldn't get good separation?
Thank you in advance.
It is a very interesting project. Are you also working in this project in new forms of neuro-scanning (e.g. using deep neural networks to analyze images and signals of new MRIs, or TMS)? We do "non-invasive neuroscience" and computational molecular neurobiology and I wonder if it is related to your project...Thanks.
Does anyone prepare big master mix batches (that means primers, buffer, magnesium chloride, dNTPs, water; so everything except for the Taq) to final concentration for genotyping purposes?
What I mean is, what happens if I prepare 1000 reactions, aliquot it and then unfreeze the reactions I need to run the genotyping PCR, add taq, and then use? Do the dNTPs or the primers get ruined if they are frozen at low concentrations?
I extracted RNA today via the trizol method. My 260/280 ratio is 1.95 while 260/230 is 1.44. I washed the pellet using isopropanol and ethanol. The cells grown and RNA extracted from were human cells.
I have somewhat shaky hands especially when anyone watches me extract RNA. However, I did not notice any debris enter my pipette tip during aspiration of the supernatant.
Would it be possible to continue and synthesize cDNA from this RNA for qPCR? Would the qPCR be accurate?
Attached: My Nano Drop
In a situation where a myelinated neuron (at any location in the nervous system) gets demyelinated, what are the changes that occur in the neuron. May be there are three phasic changes. Pre-Demyelination changes, Intra-Demyelination changes and Post-Demyelination changes. What are these changes? Can we ennumerate them.
Important: Does any change in the size/length of the neuron occur at any given instant during the process of demyelination?
I have flash frozen brains in the past using isopentane with dry ice in it but I found that some of them cracked. What is the optimal time to leave the brain in the isopentane such that it is frozen solid but the cells are still viable?
Dear sirs, I have been studying much recent literature on alpha-synucleinopathies, and I still cannot find a clear answer to the question below.
I am not so sure that a clear answers exists, but I would like to share such question with you. I am sure your expertise will help me clarify my doubts. I thank you very much in advance for your help!
Much has been said about the physiological role of alpha-synuclein. For instance, it has been demonstrated that it promotes SNARE-complex assembly, that it regulates the membrane localization of MATs, etc.
We also know that the main toxic effects in alpha-synucleinopathies are due to a gain-of-function, taking place when alpha-synuclein aggregates into soluble oligomers.
Some forms of familial PD are determined by a quantitative increase of alpha-synuclein expression (gene duplication/triplication). As a result, the physiological functions of alpha-synuclein should be exacerbated. Is it possible that some of the pathological effects of such "quantitative" forms of PD are determined NOT by a gain-of-function due to increased aggregation, BUT RATHER by an exacerbation of the physiological role of alpha-synuclein (eg.: excessive induction of SNARE-complex assembly)?
Are there any evidences in support or against such hypothesis?
Are there any evidences of reduction or exacerbation of such physiological roles, in the forms of PD caused by a qualitative mutation as well (eg.: A53T, A30P, …)?
Do such qualitative mutations alter the concentration of active alpha-synuclein species? By active I mean the alpha-synuclein species that are able to carry out the above physiological functions.
I just completed RNA extraction from brain tissue (PFC) using the RNeasy Lipid Mini kit from QIAGEN. One particular sample gave a very low concentration of 66 ng/ul RNA (in a total elution volume of 30 ul) after measured with NanoDrop.
I want to synthesize cDNA by using the QuantiTect® Reverse Transcription kit. The reaction is for 1ug RNA in a well plate of 20 ul maximum volume each well. After my calculations, the water I need to add to dilute this sample has a negative value and the volume of RNA solution to be added to the well is 15,19 ul. I am a bit puzzled as I don't seem to find any examples anywhere to help me with what I should do.
I haven't encountered this before as this particular sample was not very well isolated and the final tissue piece was very small.
Any ideas or solutions would be highly appreciated!
Thank you in advance,
As we know that, there is about 5-6 Noble prize on Drosophila melanogaster 's Brain. I am really surprise, it is really helpful to understand and solve the futuristic problem of human brain.
If is it so, then how? Could anybody explain with the good example?
I'm trying to estimate the amount of transport time needed to map out specific circuits in the spinal cord using a rabies-glycoprotein pseudotyped lentiviral vector. Does anyone know the kinetics of retrograde or anterograde transport for any lentiviral vectors? I've been trying to find information on this, ideally I'm looking for how many micrometers or millimeters/day these vectors can be transported through axons, etc.
I want to contruct a primer for assay of scavanger receptor marker MARCO , or CD163 for rat animal model. So my question is that how to consttruct a primer for this ?
I’m looking at the active zone for docking/priming/fusion between the nociceptor’s membrane and vesicles.
I would like to measure dopamine release in anaesthetised rats with continuous amperometry, what anaesthetic is most suitable for this experiment considering that I cannot use urethane? Thank you!
In my experiment, I am using isolated mice brain mitochondria from control and Ppif DKO mice and running a BN-PAGE. After transfer, I probe for anti-Ppif antibody, but the antibodies I have tried are showing non-specific binding to Ppif DKO (negative control).
Has anyone seen this before and can help me troubleshoot this?
How to prepare a single cell suspension from without disrupting the membrane integrity for ADP/ATP ratio measurement?
Dear all, I have run my tPA zymography protocol for more than 1 year without problems. Last week it stopped working. I made all new buffers and still is not working. Anyone had the same experience?
Here is the protocol: 12.6% SDS gel with casein and plasminogen (both substrates are stored in aliquotes at -20 or -80C). Running at 110V in the cold room. 2x30 min rinses with 2.5% triton-x solution, 3x10 min rinses with water. Incubation in glycine/edta buffer pH 8.3, coomassie blue R250 staining overnight, destaining, rinses in water.
Thank you in advance!
I am Working on Primary Neural Cell culture from SVZ of the Postnatal Mice Can anybody Help me to Provide Some guidance how to Precisely cut this part. and How to recognize SVZ in Brain of Postnatal mice. If you have some Videos Illustrative Diagram I could be better for me?
We have been performing a thioflavin-T dose-response curve on alpha-synuclein fibrils with very consistent results for some time now.
Recently, we received a new batch of fibrils produced by the same method as before, but by a different person as the original person has moved elsewhere. Since then we have not been able to get the same dose-response curve.
If you look at the attached graph, curve E7 was obtained using the original fibres and curves E16 1 and E16 2 using the new batch. Does anyone have any idea what might be happening?
I have had a lot of help with this experiment on here, so I hope someone can help with this question too.
Previous discussions on this experiment are below.
I am trying to decide how to place some samples that will be sequenced in a Hiseq2500. I want to be sure these are randomized and that I have enough replicates to interpret my results. I know publishing this data is tricky and reviewers always have concerns about replicates. So I want your opinion before I spend a lot of money on something that wont be published later.
I am trying to validate the use of blood transcriptomes as a proxy for brain transcriptomes. My genes of interest are found in the brain but I do not have access to many of these samples, hence I will use blood. I have collected samples like so:
Brain individual A, Blood Individual A (collection site 1)
Brain individual B, Blood Individual B (collection site 1)
Brain individual C, Blood Individual C (collection site 2)
Brain individual D, Blood Individual D (collection site 2)
Blood from 12 Individuals from collection site 1
Blood from 12 individuals from collection site 2
I can afford 3 lanes in the HiSeq2500. And because I want deep coverage I will not pool more than 12 samples per lane.
Can anyone suggest how can be used microglial cells derived from newborn mouse brain for parkinsons desease research? to study a role of alpha synuclein and exosomes?
my differentiated PC12 cells do not stick nicely to poly-d-lysine coated well plates. They are easy to wash out after a few washes by pbs or hbss solution. Any idea why and/or what to use instead? Could the adherence change while treating undifferentiated pc12 and incubating with NGF 100ng/ml?
I need to objectively do counts of neurons labeled with green and red fluorophores. I want to know what portion of those cells labeled with the green fluorophore are even dimly red above a certain intensity. The amount of red cells that are also green is not important. The fluorophores do not need to be colocalized, they only need to be in the same neuron. Is there a way to do this in ImageJ or FIJI?
Hello everyone ! I got some strange result following ischemic experiment in mice. After 3 days of MCAO/reperfusion microglia are believed to be activated and morphologically transformed to the amoeboid shape. In some MR brain section I got the expected results, however in other brain sections there was complete blank in ischemic core and no single microglia was detected. For the further clearance I have attached the iba1 stained photographs of ischemic ipsilateral hemisphere as below. Have you ever experienced such strange results?
What might be the reason that I could not detect any microglia in the ischemic core though I got the result in some other mice brain challenged with same MCAO procedure?
Can anyone explain to me why I got a large amount of myelin staining when I did IHC for Bax (Ser184) using the ABC method and DAB? I know Bax is associated with apoptotic neurons, which is what I was looking for (and did find a few nicely labeled cells), but why do I also have so much labeling of myelin?
I'm interested in investigating the nuclear role of ChAT, however all the published information I've been able to find is for human ChAT in the nucleus. Anyone know if mouse ChAT also traffics into the nucleus? It does have a predicted NLS, but I haven't found any studies specifically identifying ChAT in mouse nuclei.
I'm new to neuroscience work, so this may be a well-known thing that I'm just oblivious to
Using immunohistochemisty, I want to determine if a postsynaptic protein is juxtaposed to C-fibers in lamina II of the spinal cord superficial dorsal horn. There are plenty of marker proteins for differentiating C-fiber terminals but I was also wondering if there are any postsynaptic proteins that mark synapses formed with C-fibers- the idea being to co-localize my protein of interest with this hypothetical marker, to show it is located at a C-fiber synapse.
I will likely be using the hM4Di variant of DREADD and hope to avoid stressing the rat with an IP injection so close to a behavioral study that requires attention. IP will further be made more difficult by a headstage containing implanted electrodes that I would hope to avoid knocking around, so I am hoping to get suggestions for trying to administer CNO orally by gavage.
Sphingosine-1-phosphate1 (S1P1) regulates various molecular and cellular events in different body parts. Its expression pattern and functions are also varied dependent upon the cells types where it is expressed. Does anyone have any information regarding the effect of S1P1 in brain vasculature? Specially in ischemic condition (many paper suggests activation of S1P1 is neuroprotective in ischemic condition, however they lack the discussion on effect of S1P1 in brain vasculature). So if you have any information regarding the S1P1 signaling in brain vasculature, please do share !!!
Hello, I am trying to figure out how to induce and silence the same set of neurons in brain using dreadds. I am running into the problem that CNO activates both Gq and Gi dreadds. If I infused the Gi and Gq in the same animals, then both get activated at the same time after CNO injection. I am trying to find out the population of behavior relevant (like in open field test) inhibitory and excitatory neurons involved in regulating the behavior in the same animals by using in-vivo electrophysiology. Any help will be highly appreciated.
It is quite well established that DHPG perfused in acute hippocampal slices causes a transient decrease in synaptic transmission in hippocampal CA1 region (DHPG 100 micoM is perfused for 10 minutes in Palmer et al. 1997) but i haven't found the molecular mechanism that is proposed to underlay this short depression (i did find a lot of mechanisms by which mGluR-LTD could occur, including retrograde transmitters hypothesis)
It seems to be related to a reduction in calcium influx in pre-synaptic terminals but i don't know how this can happen upon group 1 mGluR activation induced by DHPG....
I am setting up a protocol for IF on endomembranes in hippocampal neurons. Thus far, it has been difficult to obtain crisp clear image even when using saponin permeabilization, as it seems that some structures get destroyed, especially the smaller ones. I found that in the literature MES buffer was used instead of PBS. Do you have any experience in that? What could you recommend?
I would like to know if there is a proportion in the concentration of any compound to compare iv / ip / icv administration in a mouse model.
I am wondering if anyone has used and characterized the expression of Cre in mature astrocytes vs neuronal cells using the B6.Cg-Tg(Gfap-cre)77.6Mvs/2J line of GFAP-Cre driver? http://jaxmice.jax.org/strain/024098.html
It seems that inducible lines have issues getting complete astrocyte coverage (and determining the right timing and dosage of tamoxifen) and non-inducible lines hit progenitors and neuroblasts early in development resulting in too much expression in neurons. The aforementioned line is relatively new, so I was wondering if anyone had characterized it as yet?
First I do apologize for posting my question here. I just signed up and am quite new. I just started recording fEPSP from hippocampal CA1 region and the problem is that the signal amplitude I get is very small (between 0.2-0.5 mV). I do sagittal sectioning (300 µm) in cold ASCF and incubate for 1-1:30 h at 30 degrees. I work with 2-4 month-old mice. When I put the slices under the microscope, all the layers can be easily distinguished. The fiber valley is very very small which I think can be good sign.
I have been struggling with this problem for a couple of weeks and tried many things to get bigger signal but nothing seems to work. The last thing that recently came to my mind was that maybe there is something wrong with the stimulation protocol. This is my first recording experience and I have no idea about how to make a protocol. The one that I am currently using is the one used by one of the students in other lab for recording EPSP from cortex and is apparently working perfectly for him.
Can anyone explain what each of the following is:
First level, delta level, first duration, delta duration, digital bit pattern (#3-0) & (#7-4), train rate...
Maybe I will be able to make a protocol for hippocampal fEPSP if I have a better idea about each of the above term and the best possible range they can be within.
Also, does anyone have any recommendations for the positioning of the stimulating and recording electrodes? Is there any particular area to get the best possible response?
I do appreciate anyone's help in this regard.
Thank you all in advance
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 have to use the nuclear, cytoplasmic and mitochondrial extract for my studies involving m-RNA and protein in brain isolated parts (striatum, substantia nigra, hippocampus, prefrontal cortex).
I have no much budget to purchase kits for the same like of Thermo scientific (http://www.thermofisher.com/order/catalog/product/78835?ICID=search-78835)
I wish to prepare chemical solutions for extracting them.
Kindly tell me the exact good and short reliable protocol to perform that involving ease and reproducible results...
I want to know what neuronal molecules are sulfonated in brain samples. Does anyone know how to identify and quantify all neurotransmitters and their sulfonate conjugates in a sample? or how to enrich sulfonated neurotransmitters/neurohormones? Thank you!
Can someone please give me some names of voltage sensitive proteins that are present in the voltage gated ion channels? Which have been widely studied so far?
What I have found so far was mucin, cadherin and ferritin and XIAPs.
Do anyone know anything more than this?
I'm running experiments and I need to silence the mouse DRG in culture. I'm thinking of either Lidocaine or Toxin cocktail (TTX with conotoxins). Does any one have experience in this and willing to share some information regrading protocol conc. duration etc.
I'd like to fluorescently stain for activation of GABAergic neurons in the mPFC- but have a few questions.
1) When looking at the mPFC, would it be best to use antibodies for FOS and GABA, or FOS and GAD67? What are the advantages or disadvantages to each?
2) Are there any particular antibodies you would recommend for this?
3) Any tips and tricks as far as staining goes? TBS or PBS? Dilutions?
Thank you in advance!
Frozen at -80 degrees since 2011 (no reason to believe samples were tampered with)
activated PPARy ELISA
Has anyone observed ThT-positive or -negative aggregation for Ab1-40 fragments depending on the supplier? We have and cannot understand why. The supplier of the ThT-negative fragment denies the use of any additive and the salt (TFA) is the same than that of the ThT-positive fragment purchased from another brand. Thanks in advance for your help.
There is less information available for human cortex about optimal golgi staining than for mouse or rat. But the tissue I am working with is human and has been in formalin for many years.
I am specifically looking for dendritic spines for classification purposes.
Any protocol ideas or publications for the optimal human cortex golgi staining protocols?
At the moment I am trying to isolate Purkinje cells from mouse cerebellum. I have a protocol which contains steps like 3% PFA perfusion, homogenization of the cerebellum with douncer, filtering through 100um pored nylon filters and stainings for FACS sorting. Main buffers are HBSS and PBS.
After the sorting (I sorted over Parvalbumin, although I would have GFP expressing Purkinje cells) I extracted RNA from my cells of interest by usin the RNA Kit of Macherey-Nagel. I got in total 198ng RNA with a RIN of 3.1 and bad purity. The extraction of the RNA happens 2 days after the perfusion. Until then I stored the homogenate at 4°C.
Now I am trying to shorten and improve the protocol. Does anybody has any experience with isolation of specific cell type from brain followed by extraction of RNA? I will need the RNA for RNA-Seq to compare wildtype with a knockout.
Thanks for any suggestions.
I have had this ICC working for several years and have used it for multiple experiments, but recently it has stopped working. I am now having nearly every cell in the brain labeled in every trial I do. I initially tried ordering a new lot of the primary antibody, but that made no difference. I have modified the concentration of the secondary and the time in the DAB and neither of those helped. I tried doing a no primary control to rule out nonspecific binding of the secondary and a no primary or secondary control to rule out ABC binding to endogenous biotin in the tissue. Both of those trials came out clean, with no staining, suggesting that the problem is not with the secondary or the ABC. We have tried replacing the paraformaldehyde (our brains are not perfused, so fixation is the first step of our IHC), the methanol, the H2O2, the normal goat serum, and the DAB tablets and none of these things have solved the problem. We also thought that contamination in the water may have been the problem, but I got the same result when using all solutions made with HPLC grade bottled water. Finally we thought there may have been a problem with the tissue since it had been in the -80 freezer for 2+ years and may have experienced temperature variation, but I recently included sections from a brain that was collected and sliced within the previous week and that did not solve the problem. I have run out of ideas for what could be causing the excess staining. Does anyone have suggestions on what I could try?
I am doing a Citrate synthase activity assay in synaptosomes enriched fragments from mouse brain. The synaptosomes are isolated using Syn-PER™ Synaptic Protein Extraction Reagent (87793 from thermo fisher scientific) giving functional synaptosomes. The assay works, and I find a significant difference in citrate acid activity between my two groups, however my background measuring before adding oxaloacetate to the reaction is very high - higher than the reaction after adding oxaloacetate. There is not significant difference in the activity of the enzyme in the background measurements (before the oxaloacetate is added). Does anybody know how I can solve this problem? Can I trust the measurements of my enzyme if the background is so high? Is the background important for the assay if it is “the same” (not significantly different) in all samples?
Everyone is on and about neuron image analysis in 3D (stacks of 2D images). And the reasons are legitimate (reconstruction in 3D is general, it is a standard). I wanted to ask is how much in practice scientific labs work with 3D data. It seems as most of the neuron morphology analysis in practice is actually carried out with in-vitro samples that are 2D. Do you have any reference where it would be possible to see the statistics - how many studies involve 2D and how many 3D neuron reconstructions to make conclusions in neurobiology. Would anyone have anything to add regarding their own opinion/experience?
We are trying to visualize fibers from a BDA anterograde tracer in the cortex. However, despite trying multiple different ways of staining using DAB, we are unable to get fiber staining. Any suggestions for modifications to the protocol?
The BDA tracer we are using is biotinylated, so we have tried using an ABC kit followed by DAB (which gives us no stained fibers), and we have also tried using streptavidin, and we still don't have visualization of fibers (though, this method does give us some background staining, though not fibers).
Are we missing a step somewhere? Since the BDA is biotinylated, we are under the impression that we don't need to use a primary or secondary antibody...rather we should be able to just use the HRP (with an HRP against the biotinylated species) step so that we can get a reaction when using the DAB.
Any insight/suggestions would be very helpful and appreciated! Thanks!
I need to extract proteins from mouse brain to run western blot and check the level of expression of different post-synaptic proteins.
Do I necessarily have to run synaptosomal preparation?
There are different receipts of internal solutions for acute slices (cortex, hippocampus etc ). There is no standardized receipt in the field. Some people include phosphocreatine and spermine in the internal solution, but some do not. What are their functions?
Recently I want to study neurogenesis, and try to use Edu as a cell marker for proliferation. However the edu not working very well so I decided change it to other markers, such as Ki67 or PCNA. Some friends told me ki67 only label S phase, as neuron no longer divided, I won't get Ki67/Tuj1/NeuN labeled cells in my CNS neurons culture. But wiki said Ki67 will label all cell phase except G0, which confused me a lot. Any one get Ki67 labeled neurons maker (i.e Tuj1/NeuN) yet?
I would like some clarification on focal adhesions (and stress fibers). Do proliferating cells express more or less vinculin-immunopositive focal adhesions (and phallodin-positive stress fibers)?
I'm new to this type of analysis and am unsure of the identification of everything in this image (see attached). This is a 100x image.
Is there a clear picture of a phagocytosing macrophage? Schwann cells that have detached from their myelin sheaths?
I'm wondering if I can only quantify degenerating fibers and perhaps use EM for cell analysis...
If I want to transfect some optigenetic constructor in cultured hip neurons, and then light-induce action potential in axons, what is the best option?
I want to have channelrhodopsin expression only in the subthalamic nucleus (STN) to infect specifically the neurons in the STN.
In fact, I am have a good struggling with coinjection of fluorogold &BDA in the same brain region. My protocol for this combination is quite similar to which L. Swanson reported in PNAS, 2010 (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2930585/), but with smaller injection current (+1-2 µA) and longer injection time (15-20 min). I can, however, get barely BDA labelled neurons or dendrites, but always very nice retrograde FG labelled cell bodies.
I almost always oberseve FG gets precipitated, especially up to first 5mm of glass pipette from its tip. I consider this might be a reason for a lack of BDA labeling in my case.
Any suggestions or comments? Thanks in advance!
Hexokinase I is predominant form of hexokinase family in mammalian brain. But under hypoxic conditions, there is no obvious change in its expression. In contrast, hexokinase II, which is mainly expressed in insulin-sensitive tissues and malignant tumors, experiences a robust upregulation. I think it is a norm that only when a protein's function has been compromised, then its subsitute may undergo a change to adjust to this situation.So my question is why hypoxia exposure can induce a significant uptegulation of hexokinase II whereas it has no effect on hexokinase I in brain? Is it possible that it may play some other roles in hypoxic situations? Thanks ~~
I am planning to perform a pharmacological study to assess hippocampal neurogenesis via IGF-1 receptor inhibitor. But most studies were performed in vitro or in vivo (systemic). So whether this inhibitor of IGF-1R (such as AG-1024 or picropodophyllin) can be intracranially injected by stereotaxic localization and what is the optimal dose? Thank you!