Multiphoton microscope lsm t pmt system

2A2-L1_s cells were plated in T75 flasks in DMEM supplemented with 10% bovine serum (BS) and grown to 75 to 85% confluence (Dou et al., 2011 (link)). Cells were treated with 25 mM EtOH, 5 μm filipin, or both for 1 hour. Cells were harvested in phosphate buffered saline (PBS) plus 2 mm EDTA, fixed in 4% paraformaldehyde for 30 minutes, blocked with PBS supplemented with 5% BS, and incubated with L1 mAb 5G3 (Dou et al., 2011 (link)), caveolin-1 polyclonal antibody (AB18199; Abcam), or Src polyclonal antibody (Ab47405; Abcam, Cambridge, MA) in PBS/BS at room temperature for 2 hours. Cells were washed 3 times with PBS and incubated with goat anti-mouse IgG conjugated with Alexa Fluor-488 and goat anti-rabbit IgG conjugated with Alexa Fluor 546 (Invitrogen, Grand Island, NY) in PBS/BS. Cells were washed again with PBS and fixed in paraformaldehyde. Images were captured using a Zeiss Multiphoton microscope LSM T-PMT system and Zen 2009 software from Carl Zeiss (Carl Zeiss International, Jena, Germany).