2.9. Immunofluorescence Staining

Activated microglia were identified with an anti-OX-42 antibody. Cells were fixed with paraformaldehyde (4%) for 30 min. Later, cells were permeabilized using Triton X-100 (0.3%) for 15 min. Then, cells were blocked using a goat serum blocking solution for 60 min at 37°C. Thereafter, cells were incubated with 1 : 800 dilution of anti-OX-42 antibody overnight at 4°C. Following overnight incubation, cells were incubated in the dark for 30 min with goat anti-rabbit secondary antibody (1 : 1000) or goat anti-mouse secondary antibody (1 : 1000). Cells were also counterstained with DAPI for 5 min [23]. After rinsing cells with PBS, representative fluorescence images were obtained using an EVOS® Floid® Cell Imaging Station. The fluorescence intensity was calculated by using ipwin32 software.