Fluorescent secondary antibody

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.

Cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% TritonX-100, and blocked with 10% goat serum. Primary antibodies (PAX8, 1:200, Cell Signaling Technologies; β-catenin, 1:200, BD Biosciences) were incubated overnight in 10% goat serum. Samples were probed with fluorescent secondary antibodies (Thermo Scientific) and Hoechst (Invitrogen) for one hour before washing and mounting with ProLong Gold. Images were collected on a Zeiss LSM 510 Meta Confocal microscope and images processed with ImageJ.