[Histonet] Bielschowsky's Silver Stain for Axons -

[TF]

Dear All

i have a problem in the Bielschowsky staining of axons.

I have rat brain tissues, fixed with 4% PFA or 1%PFA+1.25% Glut. With sucrose cryoprotection, cut into 20-25 um sections on a Leica cryostat.
I air dried the sections in fume hood for two days before wash in distilled water for silver staining.

I have tried several protocols, Bielschowsky staining and several modified ones. But I always get a high background - a large number of black dots appeared on the section without specific signals of fibers. I at first consider that it must be caused of contamination. With careful examination, most of these silver dots are neurons, sometimes showing the morphology of a pyramidal cell in the frontal cortex layer. The whole pattern is like nissle staining - you can visualize the neuronal body, but no fibers.

Then I tried another six slides with KMnO4 treatment, then the Oxalic acid prior to the 20% Silver nitrate incubation at 37 C. In two of the slides I observed fine fibers in the white matter - though not strong! But the other four slides are quite similar to before. 

Can anyone give me some suggestions to improve? To stregnthen the signals I observed, and to make the staining replicable?
It seems not be the problem of signal intensity, but none axons are stained in many sections. 

2009-08-18 



TF