[Histonet] Mallory's PTAH

[Tony Henwood]

I've found the Cherukian's modification of the PTAH stain to be hard to
beat.
It does not require Bouin's or Zenkers pre-treatment.

Method as follows:

 
Document Procedure:	
Phosphotungstic Acid Haematoxylin



Pathnet Code:	S PTAH M or G
Principle:	
Cherukian's modification employs an eosin solution that stains the
erythrocytes red and differentiates them from the blue fibrin.

Fixation:      10% buffered formalin.
Microtomy:     paraffin sections at 5um.

Controls:	
Use brain sections and section of muscle.  A good stain will demonstrate the
dendrites as blue where as in a bad stain they appear light grey to salmon
in colour.  Nuclei, fibrin, platelets and muscle will be blue, red cells and
collagen appear red. Muscle striations should be well defined.

Reagents: 

1.   Eosin:   - Warning: Flammable - see MSDS
           Eosin Y, water soluble (CI 145380)       0.5g
           Distilled Water                         	  10ml
           80% Ethanol                             	190ml

           Working:   10ml stock and
                   Before use add 50ul glacial acetic acid.

2.   1% Periodic Acid

3.   PTAH solution
            Haematoxylin (CI 75290)       	   0.5g
            Phosphotungstic Acid           		    10g
            Distilled water               		500ml

Dissolve solid ingredients in separate portions of the water.  Use gentle
heat for Haematoxylin.  Combine solutions when cool.  Add 0.088g potassium
permanganate to ripen.  The stain is ready to use.

 

Procedure:

1.	Dewax and hydrate sections to 80% alcohol.
2.	Place slides in eosin for 30 seconds.
3.	Wash slides in distilled water for a few seconds.
4.	Place slides in 1% periodic acid for 20 minutes.
5.	Wash slides in water for 3 minutes.
6.	Place slides in PTAH for 30-90 minutes in 60oC oven.  Check from 30
minutes on.
7.	Dehydrate, clear and mount.



Results: 
Dendrites, nuclei, fibrin, platelets and muscle - 	blue
Red blood cells and collagen - 			red.


Notes: 

Reference:	 

	1.	Cherukian, C.J., Histologic. 8(4); 105, (1977).
	2.	Luna, L., Histologic. 5(2); 66, (1975).


Tony Henwood JP, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager
The Children's Hospital at Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: 612 9845 3306
Fax: 612 9845 3318


-----Original Message-----
From: lpwenk <@t> sbcglobal.net [mailto:lpwenk <@t> sbcglobal.net] 
Sent: Friday, 13 August 2004 6:59 PM
To: UniPath IHC; histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] Mallory's PTAH


Here's what worked for us - Out of the old Ann Preece book.

Fix as usual in formalin, and process as you would for similar size tissue.

Section 4-5 um (I always vote for thinner, when working on registry
material) and place on a subbed slide. (Charged, poly-L-lysine, etc.) Helps
to hold it on through the rest of the steps. Dry in 60 degree oven for about
an hour (helps to hold the tissue on the slide during the rest of the
steps).

After deparaffinization and running down to water, post-fix in Bouin for 1
hour in a 60 degree C. oven or waterbath. Then wash in slowly running cold
water for about 10 minutes, or until all the yellow is gone. (Yes, just like
post-fixing to do a trichrome.)

Then, stain as usual with the PTAH.

Should work great, nice intense colors with very little splotching, and
sectioning should be your usual formalin quality. No mercury disposal to
worry about.

This is assuming, of course, that the PTAH solution is still good and the
procedure was followed correctly.

Now - as far as any PTAH questions on the written/computer test - the
correct answer to fixation/post-fixation for PTAH is "Zenker". As that's
what is written in all the rest of the histo books.

Hope that helps.

Peggy A. Wenk, HTL(ASCP)SLS
William Beaumont Hospital
Royal Oak, MI 48073

----- Original Message -----
From: "UniPath IHC" <ihc <@t> unipathllc.com>
To: <histonet <@t> lists.utsouthwestern.edu>
Sent: Thursday, August 12, 2004 2:13 PM
Subject: [Histonet] Mallory's PTAH


I actually already tried to use formalin-fixed muscle.  You're exactly
right, it sectioned great, but I could never get any decent staining.  I
tried post-fixing for 24 hrs. in Zenker's vs. post-fixing for 3 Hrs in
Zenker's at 56 degrees.  I tried all three protocols in Sheehan and the
protocol in Carson.  I'm at a loss for what to do next if I can't make the
Zenker fixed muscle work.  At least I've learned a lot about the stain.  :-)



Brianna Jackson, BS, QIHC

UniPath, LLC

Denver, CO



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