[Histonet] PTAH Mordant

[Tony Henwood]

Joe,

We use the Cherukian's modification and it works well. Method as follows:

Phosphotungstic Acid Haematoxylin

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).

Regards

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




-----Original Message-----
From: Joe Nocito [mailto:JNocito <@t> Pathreflab.com] 
Sent: Friday, 1 April 2005 5:27 AM
To: Histonet
Subject: [Histonet] PTAH Mordant


Hey y'all,
since we no longer have Zenkers because of the mercury, what is being used
as a mordant for the PTAH stain?  My pathologists have been hounding me all
week, but with my other net postings and replies, I just forgot.  Thanks
Histoland


Joe Nocito, BS, HT(ASCP) QIHC
Histology Manager
Pathology Reference Lab
San Antonio, TX



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