Hi Everyone,

I'm currently attempting to use Golgi-Cox staining to visualize dendritic arborization, but I'm struggling to get the full neuronal structure seen in classical Golgi-Cox staining. Has anyone come across staining such as this before?

The 200 micron slices are taken from rat brain that have been perfusion fixed with 4% PFA before overnight post-fixation. For my impregnation solution, I used 5 parts 5% Potassium Dichromate solution, 5 parts 5% Mercuric Chloride solution and a mix of 4 parts 5% Potassium chromate solution and 10 parts distilled water. Following impregnation for 1 week, I removed the brains and placed in cryoprotectant solution until the brains sunk (changing the solution every 24 hours), before carrying out the slicing. Slices were then washed 3 times in distilled water before 30 minutes of incubation in 20% ammonia. I then washed again before mounting the slices on slides and drying before sealing.

If anyone's got any idea as to what might be causing the lack of axonal/dendritic staining, as well as the bushy/blurry axonal bodies in the images, I'd greatly appreciate the advice! I have spoken to a couple of people at university and they were of the opinion that the protocol works more effectively with unfixed brains, but there are many examples in the literature of people obtaining good results with fixed brains, in addition to the fact that the issue with fixation is supposedly over-impregnation, which I don't believe is the issue. However, if you agree this is likely causing it, please let me know!

Another slight issue is that I am trying to observe the perirhinal cortex, and am finding that the cortical regions are splitting apart during the drying step - has anyone got any methods they use to avoid this? Thanks!