Have you considered doing a Sevier-Munger instead.  It is a slighty less cumbersome stain.
 
Fred

	-----Original Message-----
From: "Timothy R. Wheelock"  
Sent: Monday, June 30, 2003 3:46 PM
To: 
Subject: SILVER STAIN PROBLEMS



	Hi everyone: 

	I have been using the Bielschowsky silver stain  since 1986 on human 
brain tissue to screen for Alzheimer's, Picks, and other diseases. 
It always worked. 
Never had a problem. 

	Until about a year ago. 

	Normally one sees a  nice yellow brown macroscopic color, and clear 
senile plaques, tangles, and dystrophic neurites microscopically. 
Now, though, macroscopically, there was a greyish greenish tinge to the 
sections,  streaking of brown color across the section, with brown and 
clear bands, while microscopically, there was light staining or lack of 
almost all staining. 
Stranger still, was the fact that not all the cases turned out this way: 
some cases stained fine while others did not. 
Sometimes,  whole batches of slides would come out perfectly, and the 
next whole batch would be awful and have to be redone. 
Sometimes, cases in the same coplin jar would stain differently. 

	So, I began to systematically check things out: 
 I checked my distilled water supply (that was OK) 
 started changing my nitric acid bath regularly (never needed to 
before just topping off was fine) 
 bought all new reagents for my developer (formalin, citric acid, nitric 
acid) 
 called Fisher to see if they had changed the 10% buffered formalin we 
use to fixed our brains (no). 
  became much more fastidious about washing and rinsing the 
glasswear.(never had to before) 
  Made fresh silver nitrate every time, in new acid cleaned flasks 
(never had to do that before either) 
  
Eventually, the stains came back to almost normal. 
But not completely normal. 
It still seemed that there was a slight grayish tinge to the slides even 
when they did work well enough for both diagnostic purposes and imaging 
for our web site image archive. 

	But I noticed the other day that one or two cases seemed faint, 
especially given the fact that they had an intake diagnosis of AD. 
Our pathologist came in and complained about the stain, so I decided to 
try some other approaches: 

	1 ask for advice from people (always a good idea) 
2 buy new glassware to see if that produces a change, although I doubt 
this will help. 
3 try varying the  number of drops of the developer that I put into the 
ammonical silver solution (but why should this be connected to these 
weird effects I have experienced, when the same number of drops have 
always worked?) 
4  perhaps this may not be a silver stain problem per se....I should ask 
the pathologist if he has noticed changes with the other stains (LHE and 
Congo Red) although I do not see any and he has not complained about 
them. If it were a more general problem, maybe the LHE's would be 
slightly altered, but not enough to make a difference, while the much 
more sensitive silver stain would  show major changes. In this case, 
maybe I should get my tissue processor checked out again. ( I think this 
is a wild conjecture..the answer is probably much simpler.) 

	Frankly, I do not know what is going on. 
This should be a very straight foreword, simple stain to do. Just keep 
you glassware clean, and your developer made up properly, and that is it. 

	Anyway, does anyone have any suggestions? I would greatly appreciate them. 

	Thank you, 

	Tim Wheelock 
Harvard Brain Tissue Resource Center (Brain Bank) 
McLean Hospital 
Belmont, MA 





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