RE: PTAH Staining

From:Roberta Horner <>

	We use Meloan-Puchtler Phosphotungstic Acid Hematoxylin Method (PTAH).  No
Zenker's so no mercury.  Here is the procedure.  It is from Lee Luna's book
Roberta Horner HT
Animal Diagnostic Lab
Penn State University

Langeron's Iodine
	1 gm	Iodine (I2)
	2 gm	Potassium Iodide (KI)
	200 ml	Distilled water (H2O)
	Shelf life:	2 months
	Storage:	room temperature

5% Sodium Thiosulfate (hypo) Solution
	5 gm	Sodium Thiosulfate (Na2S2O3)
	100 ml	distilled water (H2O)
	Shelf life:	6 months
	Storage:	room temperature

Phosphotungstic Acid Hematoxylin Solution
Solution A
	1 gm	Hematoxylin (C16H14O6)
	500 ml	distilled water (H2O)
Dissolve with the aid of gentle heat but do not exceed 60 C.  Let cool for
10 minutes and add 0.1777 gm potassium permanganate (KMnO4).  Allow to stand
for at least 30 minutes.

Solution B
	20 gm	Phosphotungstic Acid (P2O524WO3nH2O)
	500 ml	distilled water (H2O)

Combine Solutions A and B and let stand for 1 hour.
	Shelf life:	indefinite
	Storage:	room temperature in a brown bottle

	1.	Deparaffinize and hydrate to distilled water.

	2.	Wash in running tap water for 10 minutes.

	3.	Treat sections with Langeron's iodine for 20 minutes.

	4.	Place in 5% sodium thiosulfate until sections are decolorized;
approximately 1 minute.

	5.	Wash in running water for 5 minutes.

	6.	Stain in PTAH solution for 2 hours in a 56-60C oven.

	7.	Dehydrate in 95% alcohol, absolute alcohol, and clear in xylene, 3
changes each.

	8.	Mount with Permount.

	"A" bands of striated muscle	blue
	Smooth muscle cells, fibrin, nuclei	blue
	Collagen, reticulum, basement membranes	blue
	Amyloid	bright red
	Elastin, membrane	dark blue


Luna, Lee G.:  Histopathologic Methods and Color Atlas of Special Stains and
Tissue Artifacts;  American Histolabs, Inc., Publications Division, 1992.
pg 436-437.

Dear HistoNet Colleagues,

     I frequently perform Mallory’s PTAH stain.  All of the techniques
I have found require mordanting in Zenker’s solution, which contains
mercury.  Because of the hazards of mercury, I would like to find an
alternative stain that will satisfy my pathologists’ needs, or find a
suitable replacement for mercury-containing Zenker’s solution.

     I would greatly appreciate any ideas you might have.  Thank you all

Best regards,
Kim Atkin HT (ASCP)

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