Immunofluorescence Imaging of BMDC Markers

BMDCs were cultured on #1.5 LabTek II eight-chambered coverslips (Nunc) for at least 30 minutes to allow firm adhesion. Cells were fixed by addition of ice-cold fixative (4% paraformaldehyde and 0.5% glutaraldehyde in PBS) and incubated for 30 min at room temperature in the dark, followed by permeabilization with 0.2% Triton X-100 in PBS for 15 min. Cells were then cultured with 10 µg/ml primary antibodies (Abs) in IF buffer (TBS plus human serum cocktail) overnight at 4°C in a humidifier chamber. Primary Abs consist of a mouse monoclonal anti-I-A^b (Biolegend), rat polyclonal anti-LAMP1 (BD Biosciences), and rabbit polyclonal anti-WASH and anti-VPS35 antibodies [7] (link), [16] (link), [42] (link) diluted in IF buffer. Following 5–6 washes in PBS, cells were incubated with secondary Abs (1∶500 dilution in imaging buffer) for 1 hr at room temperature. After 5–6 washes with PBS and addition of Hoechst 33342 nuclear stain, SlowFade Gold antifade reagent (Molecular Probes) was added to the wells. Images were obtained with an LSM-710 laser scanning confocal microscope with a 100X/1.4 Oil Plan-Aprochromat objective lens using ZEN software (Carl Zeiss). Each image represents an individual slice taken from a z-stack comprised of several slices at 0.25 µm depth.