Problem solved!
It´s the 4%PFA. I did use a fresh aliquot in one of the former tests, but is seems all our current aliquots are not working. Might be a temporal coincidence with a change of the person in charge preparing the PFA.
What is weird though, unless you try to stain f-actin either with phalloidin or sprichrome´s SPY-probe (this was included in today´s test. Same behaviour as phalloidin) - you would not realize that the PFA is not OK. Antibody stainings (e.g. tubulin, actin) and transfected Lifeact-GFP looked perfectly fine.
Today I included 4% formaldehyde in the test: perfect staining on the complete coverslip. See images below.
Because a dried coverslip was also a posibiIity suggested above, I also included this: letting the coverslip air dry after the final washing step for approx. 10 min. Here I see no difference compared to my normal mounting method where I take of the liquid as good as possible but do not wait before mounting.
These are HeLa cells transfected with Lifeact.GFP. GFP in green, Phalloindin647 in magenta.
4% PFA:
4% formaldehyde:
I am still interested if anyone has an explanation what could possibly be wrong with the PFA.
cheers,
Martin