Rabbit anti inos

Rabbit kits were anesthetized and intracardially perfused with saline followed by 10% formalin at G29, PND1 and PND5. Brains were post-fixed for 24 h and cryoprotected in 30% sucrose. 30 μm sections were cut with a cryostat and mounted onto poly-L-lysine coated slides (Sigma Aldrich, MO, USA). The sections were blocked with 5% donkey serum, incubated with goat anti-IBA1 (1:250, Abcam, MA, USA), rat anti-CD11b (1:100, Genetex, CA, USA) and mouse anti-CD45 (1:100, Bio-Rad, CA, USA), or goat anti-IBA1 (1:250, Abcam, MA, USA) and rabbit anti-iNOS (1:50, Thermo Fisher Scientific, MA, USA), followed by fluorescent secondary antibodies (1:250, Thermo Fisher Scientific, MA, USA) for 2h. For TSPO and Lectin co-staining, sections were blocked with 5% donkey serum, and incubated with goat anti-TSPO (1:500, GeneTex, CA, USA) overnight at 4°C. The sections were washed, incubated with DyLight 594 lectin (1:250, Vector Laboratory) and fluorescent secondary antibody (1:250, Thermo Fisher Scientific, MA, USA) for 2 h. Sections were washed and incubated in DAPI (1:1000) for 15 min. Immunolabelled sections were cover-slipped with Dako fluorescence mounting medium (VWR, PA, USA). All the images were captured on Zeiss LSM710 confocal microscope, keeping similar settings during image acquisition for different groups.

For immunoblot analysis, an equal amount of proteins extracted from BMDM of WT, Fcer1α^−/−, Fcer1α^−/−Nhe1^+/–, and C3^−/−mice were separated on SDS-PAGE, blotted, and detected with the following antibodies: rabbit anti-iNOS (1:1000, #PA1–036, Thermo Fisher Scientific), rat anti-Mac-2 (1:1000, #CL8942LE, Cadarlane Laboratories), mouse anti-Arg-1 (1:1000, #678802, BioLegend), rabbit anti-complement C3 (1:1000, #PA5–21349, Thermo Fisher Scientific), rabbit anti-pERK (1:1000, #4370), mouse anti-ERK antibody (1:1000, #9107), rabbit anti-pp38 (1:1000, #4631), mouse anti-p38 (1:1000, #9228) and rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (1:2000, #2118S) antibodies from Cell Signaling Technology (Danvers, MA).