[Histonet] IHC after Wright's stain

[Jan Shivers]

Hello all,

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.

Jan Shivers
UMN Vet Diag Lab