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@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 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet