Immunostaining and fluorescence microscopy

HeLa cells were fixed for 20 min in 4% formaldehyde (from depolymerized paraformaldehyde) in PBS, followed by two washes in PBS. Cells were permeabilized using 0.1% Triton X-100 in PBS for 4 min followed by two washes in PBS and blocking using 2% BSA in PBS for at least 15 min. Cells were stained by use of a primary antibody (diluted 1/300 for anti-LampI and 1/200 for anti-MBP in PBS containing 2% BSA, incubation for 1 h at room temperature) followed by three washes in PBS and incubation with the secondary antibody (diluted 1:500 in 2% BSA in PBS, incubation for 1 h, room temperature). Finally, cells were washed thrice using PBS and mounted using Mowiol. Samples were imaged on a Leica SP5 TCS II MP confocal microscope with a 63× oil immersion lens.