After 30 minutes at room temperature, cells were washed before biotinylated detection antibody solution was added to each well.
After drying the cells at room temperature, cells were fixed for 10 min with PBS containing 4% paraformaldehyde.
When these cells were shifted for further 24 h (or are lightly shaken) to the non-permissive temperature cells get separate to single cells or small clusters.
After blocking in 8% BSA in PBS for 1 hour at room temperature, cells were incubated at 4°C overnight with the primary antibodies diluted in 1% BSA.
After fixation with 1% formaldehyde (Sigma, St . Louis MO) for 20 min at room temperature, cells were collected and lysed in ice for 10 minutes.
After 4 h of incubation at room temperature, cells were washed three times with PBS/probenecid (PBS supplemented with 0.1 mM probenecid).
After blocking using bovine serum at room temperature, cells were incubated overnight with primary antibody (anti-β2AR antibody) at 4°C.
Aliqouts of 10 µl were then spotted onto glass slides and after air drying at room temperature, cells were fixed with methanol.
After fixation for 15 min at room temperature, cells were permeabilized by using 0.5% Triton in PBS for 5 min. Nuclei were stained with 4-, 6-diamidino-2-phenylindole (DAPI).
After fixation in 4% paraformaldehyde in 1x phosphate-buffered saline (PBS) for 10 min at room temperature, cells were permeabilized with 0.1% sodiumdodecyl sulfate (SDS -0.5% Triton X-100 in 1x PBSDS -0.5min [19].
Briefly, after fixation in 2,5% glutaraldeyde in 0.1 M Sodium Cacodylate buffer for 10 minutes at room temperature, cells were gently scraped from petri dishes and washed 5 minutes with 0.1 M Sodium Cacodylate buffer.
