I have a strange observation and wonder if this forum can help to shortcut my troubleshooting efforts.
I stained cells grown on a coverslip with fluorescent phalloidin (DY-647), but for some reason only the outer edge of the coverslip shows a staining (see image below). There are cells all over the coverslip (see DAPI in the second image).
Here´s the protocol in brief:
- fix with 4% PFA for 20 min
- wash 3x with PBS
- block with 5% NDS in PBSTx 0.05% (was a mistake, I intended 0.1%). approx. 30 min. [I know blocking is not necessary for phalloidin, but because others claimed the phalloidin not to work I wanted to include all steps]
- Incubate with phalloidin in PBSTx 0.1% for 30 min
- wash 3x with PBSTx 0.1 %
- mount with ProlongDiamond. (the aliquot was probably expired - can this really go bad?)
Here is an overview over the coverslip. Phalloidin-actin staining only at the edge:
Here one can see that there are cells all over the coveslip (DAPI nuclei):
→ will send this image in a second post. As a new user I am only allowed to emed 1 image per post.
Does anyone have an idea what went wrong with the phalloidin?