This seems like perhaps a technical/coverslip processing thing. I have stained thousands of slips of PFA fixed cells with phalloidin. I understand that methanol risks diminished staining, as it changes the structural pitch of the actin filaments such that the phalloidin doesn’t bind as well. (This is not to say that phalloidin won’t work on methanol fixed cells, but I’ve heard it can be problematic). It does look like there is a gradient of intensity of the phalloidin as you move toward the center…Are you inverting the slip onto a drop of phalloidin, submerging it, etc., when you perform the stain? Your cells look pretty dense, are they piled up on each other? I always used 0.1 or 0.2% triton X-100. I never included detergent with the phalloidin, but this should be ok. Somewhat unrelated - is your DAPI in the ProlongDiamond?
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