Immunofluorescence Staining Protocol for FcRn and FCGR2/CD32

The immunofluorescence (IF) staining was performed as described previously [13 (link),36 (link)]. Briefly, sections/membranes were washed three times with PBS pH 7.4 for 5 min, followed by blocking with 4% BSA, 0.5% saponin and 10% normal goat serum in PBS pH 7.4 overnight. The primary antibodies against FcRn (Pirbright Institute, Woking, UK) and against FCGR2/CD32 (Novus Biologicals/Bio-Techne GmbH, Wiesbaden, Germany) were diluted 1:100 in PBS pH 7.4 containing 4% BSA and 0.5% saponin and incubated for 24 to 48 h at 4 °C. Afterwards, the sections/membranes were washed again three times and incubated with the corresponding secondary antibody (Table 1, 1:500 diluted in PBS pH 7.4) for 2 h. Subsequently, three additional washing steps were performed. Nuclei were stained via DAPI (20 µg/mL in PBS, Thermo Fisher, Dreieich, Germany) for 10 min and washed in PBS pH 7.4. After three additional washing steps, slides were mounted with Fluoromount G mounting medium (Sigma-Aldrich, Taufkirchen, Germany).