[Histonet] PAS staining

Mahoney,Janice A Janice.Mahoney <@t> alegent.org
Wed Oct 6 12:19:36 CDT 2010

Yes, I have experienced the same thing.  Many people forget about the impact temperature has on some staining reactions.  If you keep your Schiffs and Periodic acid in the refrigerator it may take longer to stain than it would if the reagents were at room temp.  Many old procedures are written with the reagents at room temp (even the ones requiring refrigeration).
This was one of those "live and learn" situations for me.  Now temp and time are the first things I look at when a stain does not work on a known control.
Jan M

-----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 11:43 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?
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