The experiment involves uptake of a fluorescent compound and confocal imaging. Attempts to fix the cells with 4% PFA and staining 1:50 with Alexa Fluor 633-conjugated phalloidin in accordance with manufacturer's protocol resulted in weak staining. Permeabilisation with 0.1% triton x-100 (5 min) appears to have led to efflux of the fluorescent molecule, though phalloidin-staining was much more successful.
I perform phalloidin staining with cells fixed in 4% PFA and afterwards permeabilized with 1% Triton X-100 and 1% Sodium Citrate. Then I would wash the cells with PBS and finally stain them with the palloidin.
Hope it helps!
Hi! I use Texas Red-phalloidin from Life tech and permeabilize cells with PBS-Tween 20 0.2% for about 30min and use phalloidin in a 1:100 dilution, 30min incubation at RT in PBS-Tween. I've done it in HepG2 cells and it works fine, have you tried another phalloidin?? weak stainning may not only be due to permeabilization protocol but also because of the fluorophore you are using.
Hi, thank you for the suggestions. I cannot use permeabilisation here as I have noticed the fluorescence to be much weaker when the cells were permeabilised, which I suspect is due to the efflux of the fluorescent molecule.
Please try to fix with 4% PFA plus 0.1~0.2% glutaraldehyde for 30 min to prevent the efflux of the fluorescent molecule. The glutaraldehyde can effectively fix soluble proteins in the cells. Permeabilization with 0.1% Triton is necessary for F-actin staining.
- Badges
- Science topic
- Similar topics
- Methodology
- Laboratory
- Histological Techniques