Hi Anson,

The short answer is in the vast majority of cases, dendritic reconstruction and spine counts will necessitate that you fix the brain and section it, which is typically done by transcardial perfusion of a saline buffer with fixative - most commonly paraformaldehyde. If you're interested in resolving dendritic branching and spine counts, Golgi staining would suffice. There are other similar methods, such as the use of lipophilic dyes such as DiI or DiO. Either of the Golgi or the latter can give you sparse labelling of neurons which will allow you to examine neuronal morphology. However, if you're interested in total cell counts, these methods are not suitable for that, and I frankly have no advice to provide on that end - perhaps H & E staining might work for you in that case?

Best of luck,

Hy,

have a look at this website, it will lead you to a classic paper:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1244622/

I have always performed a perfusion prior to dissection, staining, and counting. It's worthy to note that fixative and concentration of fix can alter antigen access and antigen retrieval might be required. As mentioned earlier, I would suggest a DiI dye, this would not require a perfusion. 

Hai Haider F Altimimi

Thank you very much for your valuable suggestions. So I will try saline with paraformaldehyde. 

 Hai Stefan Jean-Pierre Haas 

Thank you very much

Hai Jayme Hentig 

Thank you very much for your kind information. Can I use Dil Dye for a total neuronal count? so please give any standard protocol to perform with Dill dye.

Thanks in advance

Dear Anson,

DiI is NOT specific for neurons.  It labels any cell membrane that the dye touches, so it will label all cells.  There are ways to apply small dye crystals or inject a small amount of dye in order to trace axonal projections.  It is also possible to back-label a small number of neurons (by applying the dye on the axons or in the target area) in order to investigate the morphology of their dendrites and soma.  To get the best neuronal morphology for these techniques, I suggest you perfuse the brain, since you want to look at spines. 

DiI can be applied to living tissue (it is non-toxic when dissolved in certain liquids), or to fixed tissue.  For use in fixed tissue, the brain is perfused first.  At this point, many people cut the brain into slices, just so it is easier to get the dye in place.  The dye is then applied, and then the brain is stored in fixative (usually 2% paraformaldehyde) in order to allow the dye to diffuse in the lipid membrane.  Depending on the size of the sample and the distance from dye spot to cell, this can take days or a few months.  This DiI labeling technique is also known as a "fixed fill".  In my opinion, you can get great results on fairly thick slices of fixed brain, and there are lots of examples in the literature.  I suggest that you look at some of them in order to see if they will give you the resolution that you would like for your spines.  Some of the classic papers are listed in the References section of the paper in the next paragraph. 

It is important to note that it can be risky to cryo-section tissue after it has been labeled with DiI--it can spread the dye around due to the knife tearing the tissue.  DiI is also removed during paraffin embedding.  This is one of the reasons that people have looked for a DiI-type dye that is compatible with the CLARITY technique:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5011694/ 

I do in vivo labeling with DiI and then fix the brain.  In this case, it may be better to use the DiI that is modified to be fixed by aldehyde fixation (paraformaldehyde) if it transports well in your system.

Are you able to perform immunohistochemistry?  If you wish to perform cell counts, it would probably be best to use an antibody to a marker expressed by all neurons, such as the nuclear marker NeuN.  Unfortunately, it only labels the nucleus, so it won't label the dendritic tree so that you can count spines.

Good Luck!

Jill

Dear Jill Miotke,

 yes, I am looking for IHC with NeuN.  Thank you very much for your valuable suggestion.

David M Sherry,

Thank you very much for your kind information. Is it possible to do the staining with two markers like NeuN and MAP-2 in a single microtome section (thickness 120-170 µm)?

Dear David M Sherry,

yes you are correct. Thank you very much for your valuable information.

I perfuse (4%PFA in PBS) before staining. Golgi is ok

Hai Ifukibot Usende,

Thanks for sharing.