[Histonet] H&E staining

[Rene J Buesa]

Usually inconsistency in H&E staining has two different sources: either the staining process itself, or the previous tissue processing sequence.Your staining protocol has a "standard" sequence but I would add an additional xylene and would increase the time in hemtoxylin to 7 minutes for you will differentiate ant way with the clarifier. Are you staining manually? If so the time in the clarifier should not be standard, the stained sections should remain in it until no more color leaches from the sections and should be as short as possible. Most likely your staining problems resides in the "clarifier" step and when the sections are of the "right" tissue type and the "right" thickness the staining is OK, but when they are either too thin or of tissues with less amounts of nuclei, the staining is faint.
In any even, I strongly suggest you to change your whole protocol and eliminate xylene and alcohols in the dewaxing step, and switch to a 2% aq. sol. of dishwashing soap at 90ºC (2 stations x 2 min. each) → hot distilled water (90ºC x 1 → 45ºC x 1 min) → dist. water at room temp. which will allow you not only eliminating xylene but also  finish the whole procedure sooner and much cheaper.Try it!

René 

    On Monday, February 29, 2016 6:33 AM, Charles Riley via Histonet <histonet at lists.utsouthwestern.edu> wrote:
 

 Hello all,

 We've been running into an issue lately with our H&E staining. The
hematoxylin and eosin are light on and off but the chromatin is
consistently staining light. I have changed the time in hematoxylin to
longer, used a fresh lot of both hematoxylin and our clarifier solution and
the stain is not improving.  Does anyone have any suggestions?

Here is our routine stain line procedure.
65 degree Celsius oven for 20 minutes
2x xylene for 5 mins
2x 100% alcohol for  1 minute
95% alcohol for 1 minute
DI water rinse for 1 minute
Richard Allan scientific hematoxylin for 4 minutes
DI water rinse for 2 minutes
Richard Allan Clarifier 1 for 2.5 minutes
DI water for 2 minutes
Bluing Reagent for 1 minute
DI water rinse for 2 minutes
95% alcohol for 1 minute
Eosin Y for 2 minutes
95% alcohol for 30 seconds
2x 100% alcohol for 1 minute each
2x xylene for 1 minute each
Mount and coverslip.
-- 

Charles Riley HT(ASCP)CM

Histopathology Coordinator/ Mohs

Doctors Pathology Services, Dover DE
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