[Histonet] mast cell staining in H&E

[Stephanie Weaver]

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 minutes:

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
979-845-3414
sweaver <@t> tvmdl.tamu.edu