Hi Claire,
I am afraid it does not. The PFA does partially permeabilise the cells, and I suspect that if you incubate long enough you will have some staining, but if you need a nice F-actin stain you need to permeabilise the cells.
I usually incubate the cells for 45 min at RT with the fluorescent phalloidin in a permeabiliser solution (containing either Triton 0.1% or Saponin 0.02%).
Hope this help.
Max
Hi Claire,
I (partially) agree with Massimiliano. In my experience, after PFA fixation there is partial leakage of phalloidin and that is sufficient to faintly label cells. For some reason I found that NBT-phalloidin could get into fixed, unpermeabilised cells better than with any other fluorescent label - not sure whether this is a size or charge issue. I have also found that making 4% PFA fresh ( or frrozen) from solid paraformaldeyde results in less leakage than using liquid 37% formaldehyde, probably because of contaminants and stabilizers in the liquid form. Even a small amount of triton is sufficient to permeabilise for phalloidin. Acetone fixing works well but phalloidin will not stain methanol fixed cells. Hope this helps.
You can use 0.1% triton X (very mild) for 15-20 mins at RT in order to permeabilise the cells as phalloidin will not penetrate without permeabilisation.
- Badges
- Science topic
- Similar topics
- Medicine
- Oncology
- Cancer Research
- Cancer Biology