Immunostaining was performed as described earlier [30 (link), 31 (link)]. Briefly, coverslips containing 200–300 cells/mm2 were fixed with 4 % paraformaldehyde for 15 min, followed by treatment with cold methanol (−20 °C) for 5 min and two rinses in PBS. Samples were blocked with 2 % BSA in PBS containing Tween 20 (PBST) for 30 min followed by incubation in PBST containing 1 % BSA and mouse anti-CD68 (1:200), goat anti-IBA1 (1:200), rabbit anti-iNOS (1:200), mouse anti-GFAP (1:500), mouse anti-MBP (1:200), and goat anti-MAP-2 (1:100). After three washes in PBST (15 min each), slides were further incubated with Cy2, Cy5, and DAPI (Jackson ImmunoResearch, West Grove, PA, USA). After secondary antibody incubation, coverslips were rinsed in 1X PBS, mounted on slides in Fluoromount (Sigma) and imaged using an Olympus BX41 fluorescent microscope equipped with a Hamamatsu ORCA-03G camera.
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