Adipose Tissue Whole-Mount Immunostaining

Adipose tissue whole-mount staining was performed as previously described [80 (link),81 (link)]. Briefly, isolated murine epididymal adipose tissues were fixated in 1% paraformaldehyde in 24-well plates. The tissues were then washed and blocked with a blocking buffer (PBS-0.3T with 5% normal goat serum). Blocked tissues were incubated overnight with primary MYH10 (Santa Cruz Biotechnology, Dallas, TX, USA; SC-376942) and GLUT4 (Santa Cruz Biotechnology, Dallas, TX, USA; SC-53566) antibodies. Next, the tissues were incubated with secondary antibodies, Alexa Fluor 555 anti-Mouse IgG1 (Invitrogen, Waltham, MA, USA; A-21127), and Alexa Fluor 488 anti-Mouse IgG2b (Invitrogen, Waltham, MA, USA; A-21141) and washed again before adding the Fluoroshield™ mounting medium DAPI. Images were acquired by a confocal microscope (Leica SP8; Leica, Wetzlar, Germany).