[Histonet] PAS staining

JanKeeping <@t> gmail.com JanKeeping <@t> gmail.com
Wed Oct 6 13:04:11 CDT 2010

The interesting thing about the test for Schiff reagent is that both texts  
(Freida Carson's and Sheehan Hrapchak) both specify 37-40% formaldehyde.  
Hence my confusion re; the color development

On Oct 6, 2010 3:24pm, gayle callis <gayle.callis <@t> bresnan.net> wrote:
> 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 Outdated, bad Schiffs always has the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  

> staining of basement membrane on 1 to 2 um zinc formalin or FFPE sections

> were never a problem.

> Gayle M. Callis


> -----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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