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 [email protected] _______________________________________________ Histonet mailing list [email protected] http://lists.utsouthwestern.edu/mailman/listinfo/histonet
