Immunofluorescence of Drosophila Tissues

Indirect flight muscle was dissected and fixed in 4% formaldehyde (Agar scientific; R1926) in PBS for 30 minutes, washed twice with PBS, and mounted on slides in Prolong Diamond Antifade mounting medium (Thermo Fisher Scientific; RRID:SCR_015961). Larval epidermal cells were prepared as previously described (Lee et al., 2018 (link)). Larvae were dissected in PBS and fixed in 4% formaldehyde, for 30 min, permeabilized in 0.3% Triton X-100 for 30 min, and blocked with 0.3% Triton X-100 plus 1% bovine serum albumin in PBS for 1 h at room temperature. Tissues were incubated with anti-ATP5A antibody diluted in 0.3% Triton X-100 plus 1% bovine serum albumin in PBS overnight at 4°C, rinsed three times 10 min with 0.3% Triton X-100 in PBS, and incubated with the appropriate fluorescent secondary antibodies for 2 h at room temperature. The tissues were washed twice in PBS and mounted on slides using Prolong Diamond Antifade mounting medium (Thermo Fisher Scientific). Fluorescence imaging was conducted with a Zeiss LSM 880 confocal microscope/Nikon Plan-Apochromat 63x/1.4 NA oil immersion objective. For adult eyes, images were acquired using a Leica DFC490 camera mounted on a Leica MZ6 stereomicroscope set at maximum zoom.