RE: [Histonet] PAS and how you do the staining

From:"Bartlett, Jeanine"

Gayle makes some good points:  I never reuse the Schiff's and I make fresh periodic acid every 6 months.  I also test the Schiff's as I approach the month before the expiration date.

	-----Original Message----- 
	From: on behalf of Gayle Callis 
	Sent: Mon 8/2/2004 3:59 PM 
	Subject: [Histonet] PAS and how you do the staining

	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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