[Histonet] PAS staining

[gayle callis]

I was taught to avoid aqueous fixatives when trying to retain glycogen,
soluble in NBF.   Carnoys, Gendres fluid or acid alcohol formalin are three
recommendations (Sheehan and Hrapchak Theory and Practice of
Histotechnology) followed by starting processing in 95 to 100% alcohols.
Alcoholic formalin should also work.  It could very well be your long term
storage in NBF has removed the glycogen, although basement membranes or
fungus would not be affected. This was very apparent in a study done here
where they wanted to see glycogen storage in mouse livers fixed in NBF for
over a week and routinely processed starting in 70% alcohol.  The glycogen,
for all purposes, was removed even in experimental animals who had large
quantity of glycogen in the cells (faintly stained but not what expected).


Certainly increasing time in periodic acid and Schiffs can help.  Also, one
can increase the percentage of periodic acid from 0.5% to 1%, a hint Culling
gave, as long as this is freshly made. 

However, we never use periodic acid for fungus staining, only chromic acid
since periodic acid oxidation can give false negative results with Schiffs
reagent.  This is published in J Histotechnology by Carson and Fredenburgh.


Interesting, but I still get a bright red pink color with Neutral buffered
formalin test.  I have never used concentrated 37% to 40% stock
formaldehyde, only neutral buffered formalin (fixative) which would have
fewer aldehyde groups available.  Outdated, bad Schiffs always has the
obvious purplish color with NBF. 

One thing we never allow  is return used Schiffs back into stock Schiffs.
Stock stain solutions are never contaminated with depleted, used solutions.
We date when the Schiffs was used, and not reused within the week, this is
discarded. This was particularly important with human renal biopsy work with
the renal pathologist recommending disposing of used Schiffs. Our biopsy
service did not handle a large number of biopsies in a year and this was a
way to ensure consistent PAS staining by using only Schiffs.  Successful PAS
staining of basement membrane on 1 to 2 um zinc formalin or FFPE sections
were never a problem.  

Gayle M. Callis
HTL/HT/MT(ASCP)    


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Janet
Keeping
Sent: Wednesday, October 06, 2010 10:43 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] PAS staining

I have for some time had a problem with Schiff's reagent and PAS staining.

   - I have tested each new, unopened bottle of Schiff's reagent with
   formaldehyde and always the color development was immediate, but purple,
   definately not pink.This result has been quite consistant.
   - PAS staining for glycogen using the method in *Histotechnology a self
   Instructional text,* would fail to demonstrate any glycogen in autopsy
   liver specimens.

I teach Histology at a community college and this problem has driven me
crazy for a number of years. I have tried several brands of Schiff with the
same results. Recently I obtained sheep tissues which were promptly
refrigerated and fixed after death, and I had hoped these tissues would
demonstrate glycogen. ( My thinking was that perhaps delay before autopsy
was somehow diminishing the glycogen in the specimens that I had.or that
perhaps long term NBF fixation had hampered staining.) Basement membranes
were stained with the Schiff reagent as expected despite the purple color in
the formaldehyde test. Hotchkiss Mcmanus with the same reagent also produced
beautiful staining of fungus a lovely magenta color. A search of the web
made me suspiciious when I noted that Schiff added to 37-40% formaldehyde
should produce a pink or red color, however, A spot check of formalin using
Schiff should produce a purple color. I sent an e-mail to Brian Hewlett to
ask if he could make any recommendation.

Brian was not surprised by the purple color devopment in testing, He
suggested that a large number of available aldehyde groups would be expected
to produce this color. He suggested that I increase my periodate oxidation
to 20-30 minutes and my Schiff application to 30 minutes.

This worked and I am extremely grateful!. Has anyone else had an experience
like this?
Janet
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