[Histonet] Phosphotungstic Acid Hematoxylin stain without usingZenker's

[Gayle Callis]

There is a variation of this method for CNS tissue.  

CAVEAT:You get rid of the mercury, however, dichromate solution cannot be
dumped down a drain, collect for chemically safe disposal. 

Gamble and Bancroft, 5th Edition, Theory and Practice of Histological
Techniques, 2001. 

Using naturally oxidized PTAH solution:

Dewax, and rehydrate to dist. water
Acid dichromate solution for 30  min
	10% HCl in absolute ethanol		12 ml
	3%  aqueous potassium dichromate	36 ml
	Chromate solutions must be collected, cannot be dumped down sink.

Wash in tap water
Acid permangante solution for 1 min
	0.5% potassium permanganate		50 ml	
	3% sulfuric acid			2.5ml

Wash in tap water
Bleach in 1% oxalic acid
Wash in tap water
Mallory's PTAH stain overnight
dehydrate starting in 95% alcohol, if sections are too blue, some of the
stain will be removed during dehydration.  Dehydration should be rapid to
prevent water and alcohol from removing stain. 

IF tissue has been fixed in a chromate based fixative, you can eliminate
dichromate step i.e Zenkers fixation.

If you do not have naturally oxidized PTAH, PTAH can be made with chemical
oxidation by potassium permanganate, but this solution continues to oxidize
and has a short shelf life. 

	A.	hematoxylin, 0.5g -dissolve in 100 ml distilled water

	B.	10 g phosphotungstic acid in 400 mls distilled water
	C.     Add A to B, then add 25 ml of 0.25% potassium permanganate.  Stain
can be used next day with 		optimal (they said peak) staining 	is at 7 days. 

In past, we used permanganate oxidized PTAH with success, not sure of
others experience.  If you have trouble, you should buy a naturally
oxidized PTAH from a reliable commercial source, but check with them to
make sure it is not chemically oxidized version.  PTAH, natural variety,
can last for years. 

Gayle Callis
MT,HT,HTL(ASCP)
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)