Phalloidin staining only at the edge of the coverslip

Probes & Prep
17

Unfortunately the problem is not yet solved. I promised myself to give it one more try before giving up. Did not take any pictures today but had a look at the microscope - depending on frustration level I will maybe take some images tomorrow. Probably not. I´m hoping for next week.

Today´s results in brief:

  • glycerol mounting (1):
    actin staining with an antibody (alexa488 channel) is nicely seen over the complete coverslip. Phallo647 fails. Almost nothing EXCEPT for some very nice cells in a very different z-plane (where there is no green to see). Can the glycerol RI induce such a strong abberation in z-direction? I was using a Zeiss Plan-Apochromat 10x/0,45.
  • glycerol mounting (2):
    cells transfected with Lifeact.GFP - they were glowing like crazy before fixing them. After mounting I see nothing. Also no Phallo647. Really no idea what went wrong here.
  • lifeact.GFP transfection (and ProlongDiamond mounting): very nice signal on the whole coverslip
    % when fixed with 100µl PFA top of the coveslip (18 mm diameter), there is the infamous Phallo647 ring at the edge of the coverslip.
    % when fixed with 500µl PFA in the 12well (i.e. coverslip completely immersed) I see no Phalloidin signal. Very strange. These are HeLa cells. They are not known for lacking the actin cytoskeleton, aren´t they?

Plans for the next round (more suggestions welcome):

  • so maybe it is really a drying effect that affect especially Phalloidin and not antibody staining and not GFP. Therefore: let one coverslip dry out on purpose. Any ideas at which step would be best? After fixing? After primary antibody? Before mouting? Currently I believe before mounting would be best.
  • maybe our HeLa cells grow really weird and have tons of F-actin when close to the edge of the coverslip and none when growing in the center? Therefore: break a coverslip before starting with the fixation. When half the coverslip has also an egde-staining, it can´t be the cells growing in the center are that much different (which I doubt anyways, the Lifeact.GFP looks very much the same, no matter where the cells grow).