Deparaffinization and antigen retrieval was done with commercial - ZYTOMED's OmniPrep (pH 9). Slides were washed with PBS-Tween (0.05%) and blocked (commercial - Invitrogen's CAS-Block) – 1h at RT. Anti-HLA was incubated for 1h at RT and washed with PBST three times.
Phalloidin reagent was prepared according to protocol in three dilutions: 1/250, 1/500 and 1/1000. Secondary Alexa flour 488 donkey anti rabbit was added to each reagent tube at a 1/1200 dilution. Slides were incubated for 1h at RT and then washed three times with PBST. Secondary only slices (only Secondary Alexa flour 488) were used to determine exposure time threshold.
There was no signal above background in all three Phalloidin dilutions. The picture (at the Texas Red Tfilter) was taken in slight overexposure to show the absent of staining.
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Submitted Jul 10 2017