Donna,

>From everything I've read over the histonet, I think all the bases have
been covered.  I never used the sodium metabisulfite and always had
excellent results.  I used small intestine as a control for PAS and
mucicarmine with excellent consistent staining.  I used a flood method for
PAS  ( I hated all the dishes).  This would mean the Schiff's would not be
sitting in a coplin jar.  My offer to sample the Harleco Schiff still
stands (no pressure intended).  It may help eliminate the Schiff's as the
culprit.  I think Schiff's freezing or having ice crystals would be a
problem.

Please feel free to call me at 800-222-0342 ext. 443, if you want to
brainstorm.

Rande Kline HT (ASCP)
Technical Services
EM Science




Donna Carr <dkc@odsgc.net> on 04/07/99 09:17:05 PM

To:   'Histonet' <Histonet@Pathology.swmed.edu>
cc:
Subject:  Re: PAS Problems, more info Gayle




Gayle asked for more info.
    To further elaborate on the problems we are experiencing with our
PAS stain,  I have worked in Histology for 2 years and never had any
problem with a PAS stain.  We also use the spit method for diastase.
The stain has always worked beautifully.  Usually the second the slides
are placed in the coplin jar the control turns pink.  That's why I am so
baffled at the recent problems.  The sections are not staining enough.
They are turning pink but not reacting like they normally do.  We stain
liver needle biopsies and tumors with our PAS.  We used to reuse our
reagents but have gone to using a staining rack instead of  coplin
jars.  I did try the coplin jar method without any success.

Our method is as follows:
    hydrate to di H20
    5 min periodic acid (1g/dl)
    rinse in DI
    15 min in Schiff's reagent
    2 min rinse in Sodium metabisulfate solution
    2 min rinse in Sodium metabisulfate solution
    5 to 10 min rinse in running tap water to enhance pink
    stain 4 min Hematoxylin
    dehydrate, clear, cover slip
   this is from memory and I can't remember the concentration of the
Sodium  metabisulfate?  (0.5g/100ml)

    1.  We used a different control, so this was the first suspected
culprit.  We obtained new sections from autopsy liver for controls and
ran several trials with different old control blocks and new blocks.
Not seeing any improvement in the staining.
    2.  We had problems with our reagents having ice crystals in them (
we share a fridge with chemistry) and have since moved our reagents to a
different location.  I called both companies that we get our Periodic
acid and Schiff's reagent from and both assured me that freezing would
NOT harm to the reagents.  Only to make sure that the Periodic acid was
thoroughly mixed and at room temp before using.  Concerning the Schiff's
reagent I was told that it was okay to keep at room temp and no need to
refrigerate.
   3. We are a small lab and keep reagents as long as possible.

My plan of action:
We have a new bottle of Schiff's that we are now using.  So I think the
first thing to do is to get a section of cervix to use as a control.
Secondly to use a new batch of periodic acid.  I don't believe we have
the chemicals to make our periodic acid, but have a small amount used
for our fungus stain that is the same concentration.

Any further suggestions are welcome, sorry so long winded.  Thanks again
for all the response.
        Sincerely Donna