[Histonet] mast cell staining in H&E

Laurie Reilly laurie.reilly <@t> jcu.edu.au
Tue Feb 10 17:43:27 CST 2009

Dear Stephanie and colleagues, 

We section lots of dog skin lesions and because mast cell tumours are quite
common in dogs, we routinely do a Toluidine Blue stain on all canine skin
The method is from Humason "Animal Tissue Techniques" and uses
 0.2% Toluidine Blue in 60% Ethanol for 2 minutes.
This stains canine mast cells very well but there are species differences.

Our H&E uses Mayer's Haematoxylin and an Eosin/Erythrosin B mixture, but it
does not stain the granules of mast cells. Our pathologists rely on cell
morphology if they are trying to identify mast cells in a H&E stained

Regards,   Laurie.

Mr. Laurie REILLY
School of  Veterinary and Biomedical Sciences
James Cook University
Townsville  Qld.  4811

Phone 07 4781 4468

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Stephanie
Sent: Wednesday, 11 February 2009 6:46 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] mast cell staining in H&E

Dear Histoland,

I have a dilemma that I was hoping you could help me puzzle out.  I am in a
veterinary diagnostic lab.  I have been asked to improve our H&E stain to
produce "more contrast between the blues and reds".  They would especially
like for mast cell granules to "pop out" in an H&E stain so that we have
fewer requests for special stains.  Here is our current protocol, using a
linear stainer with each station set at 1 minute so that 4 stations equals 4

1. Dry slides in forced air slide dryer at 70 C, 20 minutes
2. Xylene, 5 minutes
3. Absolute alcohol, 2 minutes
4. 95% alcohol, 1 minute
5. distilled water, 1 minute
6. Gill 3 hematoxylin from Anapath, 3 minutes
7. Running tap water, 1 minute
8. 20% glacial acetic acid in 80% reagent alcohol, 1 minute
9. Running tap water, 2 minutes
10. 80% reagent alcohol, 1 minute
11. Alcoholic Eosin Y from Anapath, 2 minutes
12. 95% reagent alcohol, 1 minute
13. Absolute alcohol, 2 minutes
14. Xylene, 3 minutes

Our tap water is alkaline enough to blue the slides adequately, but I do
intend to insert a Scott's tap water as a bluing reagent to improve
day-to-day consistency.  I have tried several changes already, including:  

-increase the time in hematoxylin
-increase the time in eosin
-variations of acid-alcohol (10% acetic acid in 80% alcohol, 10% acetic acid
in 95% alcohol, and 20% acetic acid in 95% alcohol)
-add Scott's tap water substitute for bluing
-mix and match all of the above

So far the favorite is our current protocol but replacing the acid alcohol
with the 20% acetic acid in 95% alcohol, but some pathologists find this
"too blue" and others are still not seeing mast cells distinctly enough.
Please send any and all suggestions for I have just about run out.

Thank you for your help and expertise!

Stephanie Weaver
Diagnostic Lab Supervisor--Histopathology
sweaver <@t> tvmdl.tamu.edu

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