Immunofluorescent Macrophage Staining Protocol

For immunofluorescence staining of macrophages, cells were seeded on cover slips and fixed in 4% paraformaldehyde (PFA) for 15 min at room temperature, washed with 1X PBS (3 times for 5 min each) and blocked with 1X PBS containing 5% goat normal serum and 0.3% Triton-X100 for 1 h. The cells were then incubated with primary antibody (rabbit anti- Glut 1, 1:200, 12939S, Cell Signaling) overnight at 4 °C. After that the cells were washed 3 times with PBS followed by incubation with anti-rabbit Alexa Fluor 564 (Invitrogen), conjugated secondary antibody for 1 h at room temperature. Finally the cells were washed 3 times with PBS and mounted with mounting media containing DAPI. Images were taken by fluorescence microscope (Nikon ECLIPSE TE2000-U) utilizing a 63 × oil objective lens and quantified by ImageJ software^{30 (link)}.