Imaging Macrophages and TSPO Expression

After the specified incubation period, MDM were lifted using EDTA (Sigma-Aldrich), followed by gentle scraping. Cells were washed in PBS, counted and incubated at 1 million cells/mL in 1.6% paraformaldehyde solution (Thermo-Fischer) at 4°C for 20 minutes. Using a cyto-centrifuge (Cytospin, Shandon Southern Products, Runcorn, UK), cells were spun onto slides, fixed for 15 minutes in ice cold acetone, and stored at -20°C until required. For multiple antibody immunofluorescence staining and image acquisition, protocols were adapted from Dakin et al. [34 (link)], using the primary antibodies rabbit anti-human TSPO at 1:1000 (LSbio), and mouse anti-human CD68 at 1:600 (Dako). Isotype control antibodies were a cocktail of mouse immunoglobulin G (IgG₁), and rabbit immunoglobulin fraction of serum from non-immunized rabbits, solid-phase absorbed (Dako). Immunofluorescence images were acquired on a Zeiss LSM 710 confocal microscope as described previously [34 (link)].