BMAL1 Immunofluorescence Staining in Monkeys

Immunofluorescence staining of BMAL1^+/+ and BMAL1^–/– cynomolgus monkey tissues was conducted as previously described (55 ,81–82 (link)). In short, the paraffin-embedded sections were boiled in sodium citrate buffer (pH 6.0) for antigen retrieval after deparaffination and rehydration. Then, the sections were treated with 0.4% Triton X-100 for permeabilization and 10% donkey serum for blocking. Primary antibodies were then applied at 4°C overnight. After several washes with PBS, the sections were incubated with secondary antibodies and Hoechst 33342 (Thermo Fisher Scientific). Finally, the sections were mounted with Antifade Mounting Medium (Vector Laboratories, H-1000) after several washes with PBS. The images were obtained by a ZEISS LSM900 confocal system and the mean fluorescence intensity of H3K9me3 were measured by the ImageJ software (NIH). Antibodies used in immunofluorescence staining were listed in Supplementary Table S2.