Immunofluorescence Staining of Cellular Proteins

Cells were prepared as previously described [14 (link)]. Briefly, cells were fixed and permeabilized with ice-cold 100% methanol for 4 min (FASN, mTOR, LC3B and LAMP1 staining) or 2% paraformaldehyde for 7 min followed by 5 min in PBS 0.1% TRITON X-100 (TFEB and tubulin staining) and then washed with PBS. Cells were incubated with primary antibody for 1 h at room temperature followed by washing steps with PBS containing 0.1% Tween (PBS-T). Cells were incubated with the secondary antibody (anti-rabbit, 111-605-003 (Alexa Fluor^® 647) 111-096-045 (FITC); anti-mouse, (Cy3) 115-605-003 (Alexa Fluor^® 647); Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature. Prior to mounting in fluorescence mounting medium (S3032; Dako, Switzerland) cells were washed three times with PBS-Tween. Images were acquired on an Olympus FluoView-1000 confocal microscope (Olympus, Volketswil, Switzerland) at ×63 magnification.