H and E variablility

From:Jeff Silverman

Oh boy where do I begin.
Your alcohol baths are of insufficient time, your wash after hematoxylin and
after ammonia are insufficient to properly develop the hematoxylin and
insufficient to remove ammonia residues which adversely affect eosin uptake.
Your acid alcohol should be 1% HCl in 70 % etoh and your ammonia time is way
too long and it is way too strong, only 5 seconds of a solution of 200 ml
water with maybe 10 drops of ammonia is good, make fresh daily. Wash well
after! Your eosin time is too short, even for the stronger formulations (you
did not specify the formulation for your esoin). Try this:

Xylene bath 1 ........ 3 minutes
Xylene bath 2 and 3...... 1 minute each.
100% EtOh x 2.. 1 minute each.
95% etoh x2.......1 minute each.
70%....... 10 dips (can omit).
Water 1 minute.
Hematoxylin (7- 10 minutes for a Harris, I like Gill 3 for three minutes).
Water wash 2 minutes at least.
Acid alcohol 1% HCl in 70% etoh.  5 dips.
Wash 1 minute.
Ammonia water 10 dips.
Wash two minutes.
95% etoh- ten dips
Eosin 30 seconds to 1 minute (depends on formulation)
95% etoh x 1 minute.
100% etoh x 3 1 minute.
Xylene x 3........ 30 dips each

Hope this helps doc.

Jeff Silverman HT HTL QIHC (ASCP)
Pathologist Assistant
Southside Hospital
Bay Shore NY USA 11937

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