[Histonet] cresylechtviolett (cresyl fast violet) staining problems

[Sandy Thevarkunnel]

Hi,
We just bought new cresyl fast violet from Cell point scientific to 
replace our old cresyl fast violet from Roboz which was produced in 
1995.  The new stain seems to wash away from the tissue when we put it 
in the 95% ethanol with acetic acid step rapidly, but prior to the step 
there does not seem to be any detectable differentiation during our 75% 
ETOH, 75% ETOH with acetic acid and the 95% steps that precede the 95 
with acid step.  Our protocol for making the cresyl fast violet is to 
add 2.5g to 500mL deionized H2O stir and then filter.  This recipe was 
great with the old stain but the new does not seem to be working.  We 
are staining formalin fixed human brainstems cut at 50um.
Staining protocol we use
Defatting- 3hrs in 1:1 Chloroform:Ethanol
Hydration: 7 minutes- 2x 100% ETOH, 2X 95%ETOH, 1x70%ETOH
                     10 minutes- dH2O
Staining- 4-6 minutes in CV
30 seconds dH2O
Differentiation from 75%, 75% with Acetic acid, 95%, to 95% with acetic 
acid, variable times from 1 minute to 4 minutes
Dehydration- 2x 100% ETOH for 4 minutes
Clearing- 3 changes of Xylene 2min, 1min, 30 sec.
Coverslip with Permount

Sorry for the long message I was just hoping if it wasn't the recipe it 
just might be our protocol so if anyone has any ideas I'd really 
appreciate them, I'm kind of new at this.  By the way our old CV was 
black but the solution was still purple and the new CV is green and 
solution is the same purple colour but it seems thinner.

Thank you very much,
Sandy
Boston University School of Medicine