Mouse anti human cd68 antibody

PBMCs from LTBI individuals were infected with M.tb H₃₇R_v (MOI 1:1) on glass coverslips in 24-well tissue culture plates as previously described [27 (link)]. Robust, granuloma-like structures were visually confirmed at 7 days post-infection and fixed with 4% paraformaldehyde. The structures were washed and incubated overnight at 4°C with a 30 nM solution of cFLFLFK-PEG₁₂-Cy3 diluted in HBSS+. The next day, granulomas were washed three times with PBS, blocked with 10% goat serum and 5% bovine serum albumin in PBS for 1 h, and incubated with a mouse anti-human CD68 antibody (DAKO) for 1 h. Goat anti-mouse AF647 (Invitrogen Life Technologies, Carlsbad, CA) secondary antibody was applied for 1 h after additional washes. Cell nuclei were stained with 400 ng/ml DAPI DNA stain (Molecular Probes, Carlsbad, CA). Finally, the coverslips were washed three times with distilled water and mounted on glass slides using ProLong Gold Antifade (Invitrogen Life Technologies). All incubations were performed at room temperature. Images of peptide binding and CD68 expression on granulomas were captured on an FV3000 confocal laser scanning microscope (Olympus, Tokyo, Japan) using a 60× oil DIC objective.