[Histonet] PAS staining

Tony Henwood AnthonyH <@t> chw.edu.au
Wed Oct 6 18:16:46 CDT 2010


The major problem I have encounted with the PAS stain is breakdown of
the Schiff's reagent (white precipitate). Replacement of the Schiff's
reagent usually (?always) solves this. I am not surprised that glycogen
in autopsy tissues is difficult to demonstrate. Post-mortem seems to
decrease the glycogen levels. I prefer to use bowel and kidney (mucin &
basement membranes) to initially check the solutions.

Interestingly we include glycogen-rich liver in our PAS control block
and if you audit the PAS controls from when the Schiff's bottle is
opened until it is empty, or when staining decreases, you will notice a
gradual "loss" of glycogen staining. Decrease in PAS staining of Fungi
and basement membranes is often not as apparent.


Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
the children's hospital at westmead 
Cnr Hawkesbury Road and Hainsworth Street, Westmead 
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Janet
Sent: Thursday, 7 October 2010 3:43 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

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

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