Dear Tim,
     In reading the list of things you have done, the one thing I did not see was whether you checked the silver nitrate itself. The dry stuff before it is added to any 
other reagents. Check to see if it has become contaminated in anyway. Buy a small amount of newer silver nitrate and check it against the silver nitrate you have in 
your lab. If you have access to the chemical analysis lab,  ask the people there the run it through some tests (like a scintillator).
     I hope this may be of some help.
     Sincerely,
     Paul Lockwood
6/30/03 3:46:19 PM, "Timothy R. Wheelock"  wrote:

>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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