[Histonet] PAS and how you do the staining

From:Gayle Callis

We tested commercial against in house preparation and found NO difference 
in staining quality.  We do not use kits since buying Schiffs and making up 
Periodic acid is very minimal for cost and time.  In general, Scjhiffs is 
stable but it MUST be tested. We purchase our Schiffs from Fisher, and 
store it in the refrigerator, although mfr say it is stable at Room 
temperature.  We stopped making it in house to eliminate carcinogenic dye 
weighing plus having to filter the final solution - in our estimation time 
consuming and messy and without any staining difference.   Have used 
Surgipath, Sigma, Fisher's - they all worked well.

Vibrancy of staining could be linked to more than just the Schiff's 
reagent.  After a lecture from the famous Culling, a true expert in PAS 
staining, how we did performed the PAS stain changed forever.

1)  Periodic acid is NOT stable forever, Culling advised make this 
oxidizing reagent fresh each time for PAS protocol.  His premise, this 
solution was cheap and one must insure good, consistent oxidation.  1% 
periodic acid is common, time 5 - 10 min.  Some use 0.5% for 5 - 10 
min.  Overoxidation will affect results, not a bright, pink-red.  Periodic 
acid freshness issue is also addressed in Hrapchak and Sheehans Theory and 
Practice of Histotechnology, plus they also advised weekly changes of Schiffs.

2)  Do you reuse the Schiffs reagent without testing it?  Test is 10 ml 
formaldehyde,(37%) add few drops of Schiffs, watch it turn red-purple.  If 
you have used Schiffs, it should be stored in a separate bottle labeled 
accordingly.  Do not pour it back into unused stock. Change it regularly 
(weekly per Hrapchak and Sheehan). If it has a hint of pink to it, 
discard.   Do not allow to freeze.

3) Thin 1 - 3 um sections need longer time in both oxidizer and Schiffs.

4) Culling did not bother with sulfurous acid rinses, he just rinsed 10 min 
in running tap water.  If you use sulfurous acid rinses, running tap water 
rinse afterwards will intensify color.

5) time in Schiffs reagent can vary from 10 to 30 min,  we often do 15 
and/or 20 min.  This time can vary with strength and age of Schiffs 
reagent. We made Schiff using pararosaninline hydrochloride - the pure form 
of basic fucshin mixture).   Strength could be linked to dye content of 
Basic fuchsin or pararosaniline hydrochloride or if mfr has changed 
concentration of these dyes when making up their product.

It is a good idea to go back and look at how Schiffs is made, including dye 
concentration versus what you are using commercially.
Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

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