Ab204467

Murine bladders were fixed in AntigenFix for 30 min and human bladder in 1% PFA overnight at 4 degrees. Samples were then rinsed in PBS for 5 min and transferred into 30% sucrose in PBS for 24 h. 30μm sections were permeabilized and blocked-in blocking buffer containing 0.1M TRIS, 0.1% Triton, 1% normal mouse serum, 1% normal rat serum, 1% BSA for 1h at room temperature. Staining was performed in blocking buffer for 2h at room temperature prior to washing in PBS and mounting in Fluoromount-G or Fluoromount-G with DAPI. When required, a secondary staining was performed in blocking buffer for 2h at room temperature prior to washing and mounting. Images were acquired using a TCS SP8 confocal microscope and raw images were processed using Imaris. Human antibody: anti-RORC2- PE (IC6006P, R&D); anti-CD3⁻ AF488 (UCHT1, Biolegend); anti-HLA-DR- AF647 (ab223907, Abcam) and Hoechst 33342 (29 hermofisher). Mouse antibody: anti-F4/80- AF647 (ab204467, Abcam); anti-GFP rabbit polyclonal (PABG1, Chromotek); anti-CD3⁻ AF488 (17A2, Biolegend); anti-CD3⁻ PE (145-2C11, Invitrogen) and anti-Ki67- PE (SolA15, Invitrogen). Dyes: Flash Phalloidin 488 (Bio- Legend), Hoechst 33258 (cat# 40044, Biotum), DAPI (in mounting medium, cat# 00-4959-52, Invitrogen).

Cross-sections were obtained from paraffin-embedded femoral arteries and then incubated with anti-F4/80 antibody (Abcam Japan, Chuo, Tokyo, Japan; Ab204467; RRID: AB_2810932; raised in rat; 1: 50) and anti-PDGF-B antibody (Abcam Japan, Ab23914; RRID: AB_2162180, raised in rabbit, 1:50) overnight. The antibodies were diluted with Antibody Diluent (S3022; Dako, Santa Clara, CA, USA). Nuclei were stained with 4′,6-diamidino-2-phenylindole (D1306; Thermo Fisher Scientific, Waltham, MA USA). The immunofluorescence images were obtained by a confocal microscope (BZ-X710 microscope; Keyence, Osaka, Osaka, Japan).