I'm interested in comparing AFM topography images with images coming from actin stress fibers immunostained on Hs 578Bst/HTB-125 cell line. So I’m wondering if someone could give us just on image of this kind of cell immunostained. At the moment we can’t perform this kind of experiment in our lab. Thank you so much in advance.
A simple way to stained F-actin filaments is to use a phalloidin-based fluorescent dye such as rhodamine phalloidin. Molecular Probes sell a large range of phalloidin-based dyes. I would try this first.
Thanks Damien to reply my post. I know Molecular probes is an important and reliable company too. at the moment I can't perform any kind of immunoassay or immonostaining since I asked a cell stained picture.
Dear Luca,
working with alexafluor-phalloidin or tritc-phalloidin or whatever fluorochrome-phalloidin is NOT immunostaining. It is easy, fast, very specific, just... piece of cake! Stress fibers will be brilliantly revealed and you don't have to wait for any picture to get started. You only need a step of mild fixation (2-4% PFA in the relevant buffer), a mild extraction step with DMSO+Triton and then a staining step. You only have to standardise the duration and temp of each step to your cell type. Any result that you get is credible. In case you think that stress fibers are poorly preserved, then try to pre-stabilize actin by a 20min RT step in 300μM MBS (maleimido-benzoyl-hydroxy-succinimide ester), then proceed directly with fixation etc.
Good luck!
Hi Emmanuel, I'm so sorry to thank you about your reply in so late time. Unfortunately I didn't check research gate for long time. I'll pick up your suggestions for sure. Have a great day
Luca
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