Immunohistochemistry and Imaging

Tissue was harvested at designated times (as described). Prior to freezing in OCT blocks, tissue was fixed in 4% paraformaldehyde in PBS at 4°C for 8–12 hr, followed by 30% sucrose in PBS overnight. Tissue in OCT blocks was cut into 7- to 10-μm sections with a freezing microtome (Cryostat, Leica). For antibody labeling, primary labeling was performed overnight at 4°C in PBS supplemented with 10% serum from the species in which the secondary antibody was raised and 0.05% Tween 20. Secondary labeling was performed for 60 min at room temperature. Nuclei were stained using Hoechst 33258 (Cat. No. H3569; Thermo Fisher). For imaging, samples were mounted with ProLong Diamond Antifade Mountant (Cat. No. 36961; Thermo Fisher) and covered with coverslips. Representative images were taken using a Zeiss Axioskop2 Plus microscope or a Zeiss LSM 700 confocal microscope. Images were analyzed using ImageJ (http://fiji.sc/) software. Image tiles were acquired using Zeiss ApoTome.2, and cell counting was performed using Imaris software. All antibodies used for FACS and immunohistochemistry are listed in Table S2.