[Histonet] Re: H&E staining question

Teri Johnson TJohnson <@t> gnf.org
Tue Jul 2 12:24:08 CDT 2013

Hi Brett,

I agree with the others, the storage in 70% ETOH is likely not the cause of the washed out appearance of the staining.

*       Did the lab that did the H&E staining run a daily control? How did that look? Perhaps the tap water they are using went a little funny? Perhaps the depar xylene needs refreshing? The hematoxylin is used up? If all the other H&Es done that day look good, then look further.

*       Look at the fixative, it is possible to get bad batches of that. It is also possible to cram a lot of tissue into a small space resulting in inadequate fixation. Or perhaps it was bloody and not changed to fresh fixative? Was one sample fixed at room temperature and the other fixed at 4 degrees C? Was the person doing the necropsy the same among animal batches?

*       Two things might help get better hematoxylin staining - I recall a journal article about using antigen retrieval pretreatment to improve H&E staining on samples that had been stored in fixative for a prolonged period of time. That might help.  Years ago,  I also noticed that the hematoxylin counterstained PAS slides looked better than our H&Es, so we put a bucket of 0.5% periodic acid as an oxidation step before hematoxylin. It needs to be rinsed well so there is no periodic acid carryover (that'll kill your hematoxylin!), but that might improve your staining.

If you get this figured out, please let us know the cause and fix.

Teri Johnson
Manager, Histology
GNF - San Diego, CA

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