Myh10

Immunofluorescence staining was performed as described in Mor-Yosef Moldovan et al. [6 (link)]. Briefly, the cells were fixed with a 4% paraformaldehyde solution, permeabilized with 0.5% Triton in 1% TBST, and then blocked with a blocking solution (1% TBST containing 1–2% normal goat serum and 1% BSA). Next, the cells 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, washed, and incubated with secondary antibodies, Cy3-anti-mouse (Jackson ImmunoResearch Laboratories, West Grove, PA, USA; 115-165-003), 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) for one additional hour. F-actin filaments were stained with fluorescein isothiocyanate-labeled phalloidin (Sigma-Aldrich, St. Louis, MO, USA; P5282). The stained coverslips were mounted on slides with Fluoroshield™ mounting medium containing 4′, 6-diamidino-2-phenylindole (DAPI). Images were acquired by a confocal microscope (Leica SP8; Leica, Wetzlar, Germany) and a fluorescence microscope (Eclipse Ci; Nikon, Tokyo, Japan).

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).