[Histonet] Karnovsky-Roots turning green

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Dear Community.
I am currently staining whole mount preparations from Paraformaldehyde-Perfusion fixed tissues for Acetylcholineesterasis using the method of Karnivsky and Roots, which is well established in our lab. I am floating the quite thick tissue pieces (up to 1 mm) for 2 hours at 37degrees and then overnight at room temperature in the solution. Unfortunately, some of the tissue turnes into a green (old copper roof-like) colour, with some nerves turning white instead of brown and some areas not stained at all. The solution also seems to show a poor penetration through the tissue. Has anibody suggestions to improve the tissue penetration of the solution. And has anibody experienced the same colour chages and maybe knows how to avoid that? As the tissue is stored in PBS, could it be some Copper-Salt forming??
Any suggestions welcome.
 
Martina Pirker
Children's Research Centre, Crumlin
Dublin 12
Ireland

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