I am currently staining brain tissue (perfused 4% paraformaldehyde) that has been cut in a cryostat at 50 microns and I am staining for NeuN using DAB tablets with Cobalt from Sigma.

 

The stain works perfectly. I am using a mounting medium that is glycerol based (PVA-glycerol). This mounting medium also works perfectly as it can be quickly applied to the sections after they have been mounted on a slide which perserves the thickness of the sections (I am interested in doing stereology) and it polymerizes overnight so that nail polish is not necessary to keep the coverlip on. (I have used the same medium with fluorescent tissue except DABCO was added)

 

But I am noticing that over a number of days that may sections which have neurons stained black, turn brown and pale. Is this fading a product of the mounting medium? Does the fading progress to where the precipitate can no longer be seen? Has anyone used aqueous mounting mediums with DAB and had fading? It was my understanding that DAB didn't fade (or at least not too much).

 

Comments? Suggestions? Please help me put my concerns to rest.