Dapi mounting medium h 1500

Inguinal fat pads were dissected, embedded in optimal cutting temperature compound (Sakura, Torrance, California), and cryosectioned at 30 μm thickness. For immunofluorescent staining, sections were incubated with the following primary antibodies: anti-CD31, anti-CD34, anti-Gli1, anti-alpha smooth muscle actin (SMA), anti-beta III tubulin (TUBB3), anti-osteocalcin (OCN), or anti-perilipin 1 (see antibody details in Supplementary Table S1). Sections were washed with phosphate-buffered saline (PBS) three times, 10 minutes each. All sections were blocked with 5% goat serum in PBS for 1 hour at 25 C; antigen retrieval was by trypsin enzymatic antigen retrieval solution (ab970; Abcam, Cambridge, Massachusetts). Primary antibodies were added to each section at their respective dilutions and incubated at 25 C for 3 hours or overnight at 4 C. Next, Alexa Fluor 647-conjugated secondary antibodies (1:200) were used. Sections were counterstained with DAPI mounting medium (H-1500, Vector laboratories, Burlingame, California). Unless otherwise noted, all immunohistochemistry stains were examined under a Zeiss 800 confocal microscope (Zeiss, Thornwood, New York).