Anti rat alexa 647

CAD and/or HeLa cells and cocultures (CAD-CAD and CAD-HeLa) were fixed with 4% paraformaldehyde (PFA) for 20 minutes at RT. After 3 washes with PBS, cells were permeabilized with 0.1% Triton X-100 in PBS for 3 minutes at RT. Cells were then washed with PBS, and nonspecific antibody binding sites were blocked by using 10% of goat serum (GS) in PBS for 30 minutes. Cells were then incubated with primary rat monoclonal anti-mouse LAMP1 (1D4B) or anti-human LAMP1 (H4A3) primary antibodies with the dilution of 1/200 in blocking solution for 1 hour. Antibodies were purchased from Developmental Studies Hybridoma Bank (DSHB, Iowa City, Iowa, USA). Cells were then washed 3 times with PBS and incubated for secondary antibodies (anti-rat Alexa-647 or anti-rat Alexa-488 purchased from Invitrogen) diluted 1:500 in blocking solution for 2 hours at RT. After 3 washes with PBS, cells were further stained with HCS CellMask Blue (Invitrogen) for 15 minutes at RT (diluted 1:5,000 in PBS). Cells were then washed 3 times with PBS and further stained for DAPI (diluted 1:5,000 in PBS) for 5 minutes. Finally, cells were washed and mounted with Aqua-Poly/Mount (Polysciences, Warrington, Pennsylvania, USA). Image acquisitions were performed by confocal microscopy with identical settings.