Not sure if the observed pattern is real HEF1 staining or background HEF1 is supposed to localize to focal adhesions but don't see much co-localization with actin here. I also got the same results with a 4% PFA followed by a 100% methanol fix.
Image shows mouse embryonic stem cells stained for DNA (DAPI, blue), actin (phalloidin, green) and HEF1 (red).
We have had no previous complaints for this antibody.
We have no data on the PFA fixation length and permeabilization step from the customer.
Protocol tips were given to the customer:
1. Incubate primary antibody overnight
2. Block with donkey serum
The above should help obtain specific stronger staining.
Prof. Reiter Lab
Submitted Jul 07 2010
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