RE: [Histonet] Serum Fiber in Blood

From:"Tony Henwood"

Have you tried this PTAH that does not require mercury:

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 5ým.

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.	Stock Eosin:   - Warning: Flammable - see MSDS
           Eosin Y, water soluble (CI 45380)       0.5g
           Distilled Water                         	  10ml
           80% Ethanol                             	190ml 

2.	Working Eosin Solution:
Stock Eosin		10ml
Before use add 50ul glacial acetic acid.

3.	1% Periodic Acid

4.	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 working eosin solution 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 solution 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.


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: histonet-bounces@lists.utsouthwestern.edu [mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Henry, Charlene
Sent: Wednesday, 1 November 2006 2:51 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Serum Fiber in Blood


Our mouse pathologist has previously ordered the PTAH stain for blood serum fiber in mouse tissues; however we plan to retire this stain because of the mercury used in the stain. This is the last place in the lab that requires mercury. My question is: do any of you know of another special stain or antibody that will detect blood serum fiber in FFPE mouse tissue? Thanks, Charlene
 
 
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