Multicolor Immunostaining of Adipose Tissue

The following primary antibodies were used: rat anti-endomuchin antibody (V. 7C7) (Santa Cruz Biotechnology, CA, USA) (1:100), goat anti-mouse leptin receptor (R&D SYSTEMS, Minneapolis, MN, USA) (1:100), goat anti-mouse-CD31antibody coupled to Alexa Fluor (AF) 488 (BD Biosciences, San Jose, CA, USA) (1:50), rat anti-mouse osteocalcin antibody (R21C-01A) (1:200) (Takara, Shiga, Japan), rat anti-CD45 antibody coupled to allophycocyanine (APC) (30-F11) (1:500) and rat anti-Ter119 (TER-119) antibody coupled to APC (1:500) (both from Thermo Fisher Scientific, Waltham, MA, USA), rat anti-Ly-6A/E (Sca-1) antibody coupled to AF 488 (D7) (1:500), hamster anti-CD29 antibody coupled to AF 488 (HMβ1-1) (1:500), rat anti-CD90.2 antibody coupled to AF 488 (30-H12) (1:500), rat anti-CD146 antibody coupled to AF 488 (ME-9F1), isotype control rat IgG2a, κ coupled to AF 488 (RTK2758) (1:500) and isotype control hamster IgG coupled to AF 488 (HTK888) (1:500) (all from BioLegend, San Diego, CA, USA), rabbit anti-adiponectin antibody (1:10) (Novus Biologicals, Centennial, CO, USA), rabbit anti-osterix antibody (1:100) (Abcam, Cambridge, UK) and normal rabbit IgG (60024B) (R&D SYSTEMS).
Donkey anti-rat coupled to AF 647 (1:1000) (Abcam), donkey anti-rabbit coupled to AF 488 (1:1000) and donkey anti-goat coupled to AF 647 (1:1000) (both from Thermo Fisher Scientific) were used as the secondary antibodies for immunostaining. Nuclei were stained using Hoechst 33342 (Thermo Fisher Scientific). Dissection, sectioning, and staining for each experiment were always performed at the same time and conditions.