Silver stain in fixed brain tissue

[jfrasca]

Hello All,

   I have found many silver stain protocols for looking at neurodegeneration but two things are unclear:
1) Most protocols use paraffin imbedded tissue, will 4% paraformaldehyde (PFA) fixed mouse brain tissue work just fine or is the type of fixation I used a problem. I have to use that type of fixation because the brains are already sectioned and fixed in that manner.
2) Will the Bielschowsky Silver Stain Protocol for Axons, Senile Plaques and Neurofibrillary Tangles in Alzheimer's disease that is listed under IHC world protocols work for this? I know it says plaques will be visible but I assume degenerating neurons should also be visible with this protocol. I believe that is correct but I want to check with people that have used it. If not, does someone have a better protocol that will work in PFA fixed mouse brain tissue?
     I have heard from many people it is a difficult stain but the protocol doesnt look too bad. Can i use a neutral red counterstain on this as well? Will that be disrupted by the type of fixation I used?
     Thank you in advance for your responses!

[CanuckPhD]

PFA is fine to use with the Bielschowsky stain and neutral red is fine as a counter stain (though we usually don't perform one). This stain will show all axons, not just degenerating ones. You will have to rely on morphology (swelling, spheroids and torsion) to tell the difference between the normal axons and dystrophic ones. If you are looking for a stain for degenerating axons, have you considered Fluoro Jade.
The staining is tricky but it is not too bad. Be prepared to give yourself a couple days to get it working in your hands and don't give up after a few less then sucessful trials.

[jfrasca]

Thank you for the response. I have considered fluorojade but I was told it was time sensitive...but I am not sure about that. I am looking weeks after the initial insult. Also, I have immune cells that I am sure are apoptosising over time in this tissue so fluorojade would possibly be a false positive.
    The reason I asked the first question is that the neuroscience associates, inc calender claims that the "amino cupric silver method of deOlmos reveals disintegrative degeneration of cell bodies, dendrites, axons and terminals" and they use neutral red to show normal neurons. I thought the stain was a simple cupric silver stain, am I wrong? I am sure I am not understanding this right but if I described the wrong stain and the above description is another type of stain please let me know. Typically I would be skeptical of such a cool sounding stain solving all my problems but if anyone has any more information on that subject I would really appreciate the input. Thank you in advance!
Also, here is a link to the stain on their site:
http://www.neuroscienceassociates.com/Stains/silver_degen.htm

[CanuckPhD]

I have not performed the amino cupric silver method as it can only be performed on thick sections (as far as I know). We have contracted Neuroscience Associates to perform the staining for us in the past and the results are good. Neutral red is a generic nuclear stain and will reveal glia, invading inflammatory cells etc. in addition to neurons. But the morphology of the cell usually allows identification as a neuron.

The Bielschowsky's silver stain is widely used on 10um (or even 7um) thick FFPE tissues in both research and clinical labs. It can identify dystrophic neurons associated with Alzheimer's plaques or loss of axons in Multiple Sclerosis patients, as well as their respective animal models.