[Histonet] Phosphotungstic Acid Hematoxylin stain without usi ng Zenker's

[Tony Henwood]

Dear Boswell,

The following Cherukian's modification works very well.

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

http://www.histosearch.com/homepages/TonyHenwood/default.html
http://us.geocities.com/tonyhenwoodau/index.html



Document Procedure:	
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).

-----Original Message-----
From: Boswell [mailto:kcboswell <@t> grandecom.net]
Sent: Saturday, 20 March 2004 7:30 AM
To: Histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Phosphotungstic Acid Hematoxylin stain without using
Zenker's


Does anyone have a procedure for Phosphotungstic Acid Hematoxylin stain
without using Zenker's.  We have looked everywhere and are unable to find
it.  Thank you.
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