I have two stains I would like to use - namely, phalloidin, for staining the actin filaments and also DAPI for staining their nucleus. However, i cannot really find any protocols for which uses two stains on a single sample on the glass slide.
Kindly advise. Thank you.
Phalloidin mammalian cells: wash cells 3xPBS and fixation for 20 min in 4%PFA in PBS. premeablize cells with 1% triton PBS for 3-5 min wash with PBS and block cells in PBS+1%BSA for 1 hour. Add Phalloindin in BSA/PBS solution wash cells 2x PBS.
For DAPI: inculation for 20 min in PBS is enough and then wash and mount.
Good luck!
Dear Ms Sonia,
Thank you for your suggestion!
Is it possible to do both staining on the same cells concurrently?
Hi Justin,
Yes you can stain the same cells with both at the same time.
Sonia's protocol is good, but I will add that if you are only staining for phalloidin and DAPI, then it is unnecessary to block. Simply fix/perm the cells, add the phalloidin and DAPI (simultaneously), let sit at room temperature for 20-30 minutes, wash twice and mount.
Edit: To add, the reason you don't need to block is because phalloidin is not a protein and it won't have non-specific interactions (like an antibody would). It is a toxin with extremely high specificity for F-actin.
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