Immunofluorescence Staining of Macrophages

Extracted macrophages were collected by centrifugation at 500 g for 5 min at room temperature. The cell pellet was resuspended in a small volume of Phospho-buffered saline (PBS) and smeared on a cover slip. The cell suspension was left to dry before cells were fixed with 4% paraformaldehyde in 0.1M Phosphate Buffer for 20 min at room temperature. Cells were washed 3 times in 0.1M PBS and permeabilized in 0.5% Triton-X 100 in PBS. Cells were blocked for 1 hr at room temperature in 20% Fetal Bovine Serum +0.25% Triton X-100 in PBS. Primary antibodies were diluted in blocking buffer: anti-HA (Roche, Basel, Switzerland) 1:50, anti-Golgin 84, 1:25, anti-Calnexin 99a 1:25, anti-Hrs.8.2 1:25 or anti-Rab7 1:25 all from DSHB (Riedel et al., 2016 (link)), and incubated for 1 hr at room temperature. Cells were then washed 5 times in blocking buffer. Secondary antibodies were diluted in blocking buffer: anti-rat 633 1:300, anti-mouse 488 1:300 (both from ThermoFisher Scientific, Waltham, Massachusetts, USA). Secondary antibodies were incubated for 1 hr at room temperature. Cells were washed 5 times in PBS + 0.1% Triton X-100 and mounted in VectaShield + DAPI (LifeTechnologies, Carlsbad, USA) utilized at 1:75.