I use phalloidin-iFluor 488 from Abcam(ab176753) to stain F-actin in cortical neuron cells. But it doesn't work.
I think it should be easier to stain F-actin. What should I take care when I do the experiment? Does its binding with F-actin is sensitive to temperature or fixation?
Hi
I never had problems with phalloidin stainings in primary hippocampal cultures. Could you give us some specifics on how you fixed and prepared your stainings? I roughly follow the instructions and never had problems. We usually use AlexaFluor488 conjugated phalloidin. With a quick search I could not find any significant difference bewteen the two conjugates taht would explain your failed staining.
Hi, Max Adrian
I fixed cells with 4% PFA for 30min, then I use 0.5% Trion-100 to permealize for 30min , then followed by 5%BSA blocking for 30min, finally I add 1:1000 diluted Phalloidin to cells. First time I put cells in 4°, next time I put them in 60°(hope to improve binding). Both of them failed. I guess Phalloidin only work in RT?
Hi Yang,
In cultured neurons Phalloidin should work beautifully, given that all neurons are loaded with F-actin. What do you mean when you say it does not work? Do you mean too much back ground or there is no labelling at all when you look with the right set of fluorescence filters? If you seeing too much background that may require you to reduce the number of cells.
Hi Yang!
I guess 1:1000 dilution is the problem, try lower dilution. I typically use 1:150 (phalloidin A468 from Cytoskeleton, http://www.cytoskeleton.com/phdr1), this might sound too much but I follow datasheet from Cytoskeleton and it always works. Check the abcam datasheets or references if dilution is an issue. One other reason could be storage conditions, always store the compound in small aliquots at -80 deg. Best Praveen
Hi
your protocol sounds good. I incubate phalloidin for 1h ar RT or overnight at 4 degrees. You could increase the concentration of phalloidin. We usually use it 1:100 or, depending on the conjugated dye, even 1:50. I dissolved the phalloidin in MeOH as specified in the instructions.
Phalloidin is also sensitive to preated freeze-thaw cycles. You may consider using a fresh aliquot and make sure to always vortex the phalloidin dye before use throughly!
Hi
After go through your protocol, I think there are two steps which can be modified. The first one is to shorten the permealization time to 10-15min, since phalloidin will detect filamentous actin. The second one is to increase the concentration of phalloidin. I used to invitrogen phalloidin, and diluted as 1:40-1:50, which works very fine. I don't know the abcam one can be diluted as 1:1000.
As mentioned before permeabilization time can be decreased (0,1% Triton for 5 min is totally enough) and increasing phalloidin 1:50-200 for 1h at room temperature should make it work.
Regarding phalloidin sensitivity to fixation, Phalloidin is sensitive to methanol fixation or buffers with high methanol content. It won't decorate f-actin in cells fixed with pure methanol.
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