[Histonet] IHC after Wright's stain
I apologize if this comes across as an elementary histo question, but I'm not a histotechnologist myself, so am coming to you experts.
I stain blood and cytology smears on a rare occasion with the IHC method, but have never destained and then IHC-stained the same smear. This week I attempted to do cytokeratin IHC on a nasal smear that was formerly stained with Wright's stain. It was destained with acid alcohol by the Histo lab here, and then I hand-stained it for cytokeratin using the procedure I normally do with all other smears and cytospins. The positive control CK worked fine (though admittedly it was an enzyme-digested FFPE slide and not a destained Wright's smear), but the smear (with obvious epithelial cells in it) was totally negative. Twice.
Is there some constituent/step of a Wright's stain or procedure that would totally negate immunoreactivity for IHC? I assume I did not need to enzyme-digest the smear, since there was no aldehyde-type fixative used on it. Or, was the problem in the destaining reagents?
The smear did go through a 100 second methanol step during the Wright's stain, but that's all the fixation it got before coming to my lab. I gave it an extra shot of 75% acetone/25% ethanol for 8 minutes, then a thorough air-drying before starting the IHC run (normally I give my smears a 10' fix in acet/etoh). Would this combination of alcohols have diminished any reactivity? Would the delay in fixing cause a 100% lack of cytokeratin staining?
Thanks in advance for any input.
UMN Vet Diag Lab
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