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 Outdated, bad Schiffs always has the obvious purplish color with NBF.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-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Janet Keeping Sent: Wednesday, October 06, 2010 10:43 AM To: histonet@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 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet __________ Information from ESET Smart Security, version of virus signature database 5510 (20101006) __________ The message was checked by ESET Smart Security. http://www.eset.com __________ Information from ESET Smart Security, version of virus signature database 5510 (20101006) __________ The message was checked by ESET Smart Security. http://www.eset.com __________ Information from ESET Smart Security, version of virus signature database 5510 (20101006) __________ The message was checked by ESET Smart Security. http://www.eset.com _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet