S302380

Human and frog ovaries were fixed in 4% PFA in PBS overnight at 4 °C, washed, embedded in paraffin blocks and cut into 5 µM sections. After deparaffinization, antigen retrieval was performed by heating the slides for 15 min in 10 mM sodium citrate at pH 6. Sections were blocked and permeabilized in 3% BSA, 0.05% Tween-20 and 0.05% Triton X-100 for 1 h at room temperature. Sections were incubated overnight at 4 °C in the presence of primary antibodies (1:100): anti-ATP5A1 (Abcam, ab14748) and anti-HSPE1 (Thermo, PA5-30428); then 2 h at room temperature with secondary antibodies (1:500). Antibodies and dyes used were as follows: goat anti-rabbit Alexa488 or Alexa555 (1:500, Thermo, A-11008, A-21428), goat anti-mouse Alexa647 (Thermo, A21236) and Hoechst dye (1:500, Abcam, ab145597). A droplet of mounting medium (Agilent, S302380) was added onto the section before imaging using the LAS X software (Leica, v3.5.5.19976) in a Leica TCS SP8 microscope equipped with 40× (NA 1.30, Leica 506358) and 63× (NA 1.40, Leica 506350) objectives.

Cross‐sections (10 µm) were cut from the muscle mid‐belly in a cryostat at −20°C and adhered to SuperFrost Plus slides. Sections were air‐dried and then stained with hematoxylin and eosin (H & E). Slides for immune cell staining were fixed in acetone at −20°C and then air‐dried. Satellite cell staining slides were fixed in 4% paraformaldehyde (PFA), quenched with hydrogen peroxide, and antigen retrieval performed. Unfixed tissue sections were used for muscle fiber type staining. Prepared slides were blocked in 10% normal goat serum (Invitrogen 10000C) or Mouse on Mouse (M.O.M) blocking reagent (Vector Laboratories, MKB‐2213) as appropriate prior to overnight incubation at 4°C with primary antibodies. The following day, slides were incubated with Alexa Fluor conjugated secondary antibodies and mounted using Fluorescence Mounting Medium (Agilent Dako, S302380). Fluorescent images were captured using a Nikon A1 confocal microscope.

HeLa cells were reseeded 24 h post-transfection on 12 mm glass coverslips (Fisher Scientific). The next day, cells are fixed for 20 min with 4% EM-grade paraformaldehyde (PFA; Electron Microscopy Sciences 15710) and 4% sucrose in PBS at room temperature and washed three times in PBS.
To visualize the plasma membrane, cells were incubated with wheat germ agglutinin (WGA) conjugated with Alexa Fluor 594 (1:200, Invitrogen w11262) for 10 min at room temperature and washed twice with PBS. Afterwards, the nuclei were stained with Hoechst 33342 (1:2,000; Invitrogen H3570) for 10 min at room temperature and washed twice with PBS. Coverslips were mounted using fluorescent mounting medium (Dako S302380) and stored at 4 °C before being subjected to fluorescence microscopy.
To visualize the ER, fixed cells were permeabilized with 0.5% Triton-X in PBS for 5 min. After washing with PBS, coverslips were blocked with 3% BSA in PBS for 1 h at room temperature. Next, cells were incubated for 2 h with a primary chicken anti-calreticulin antibody as a marker for the ER (1:500, Ab14234, Abcam, USA), followed by three washes with PBS and staining with an Alexa Fluor 594® conjugated goat anti-chicken IgY secondary antibody (1:500, A-11042, Thermo Fisher Scientific, USA) for 1 h. Nuclear staining and mounting were performed as described above.

Mice were fasted for 4 hr and subjected to an oral gavage of BODIPY 500/510 C₁, C₁₂ fatty acids (2 μg/g BDW, #D3823; Molecular Probes). 3 hrs later, mice were euthanized and subcutaneous WAT was dissected. Tissues were immediately frozen in liquid nitrogen and stored at −80°C. Tissue pieces <2 mm were excised, mounted with fluorescence mounting medium (#S302380; Dako) on microscope slides with cover glasses, and examined for fluorescence with argon-ion laser excitation at 488 nm on a Leica TCS SP5 confocal microscope.