Lsm 880 confocal microscope

When immunolabeling cells for live‐cell imaging, cells were washed with phosphate‐buffered saline (PBS) and incubated with primary antibody on ice for 1 h. They were then washed three times with ice‐cold PBS (containing 0.5% bovine serum albumin; BSA) and incubated with the appropriate fluorescence‐conjugated secondary antibody on ice for 1 h. When immunolabeling fixed cells, cells were fixed with 4% Paraformaldehyde, and then with PBS containing 0.1% Triton X‐100 for 15 min. The permeabilized cells were washed with PBS, blocked with PBS containing 5% BSA for 1 h at room temperature, and then incubated with primary antibody at 4 °C overnight. The cells were washed again and incubated with the appropriate fluorescence‐conjugated secondary antibody for 1 h at room temperature. After another round of washing, the cells were mounted with ProLong Diamond Antifade mountant (Thermo Fisher, P36970). Images were acquired with either a Zeiss LSM 880 confocal microscope or a Nikon A1HD25 High‐Speed and Large‐Field of View Confocal Microscope, after which they were analyzed with the ZEISS ZEN microscopy software or the Nikon NIS Elements AR software, respectively.

The mouse dorsal skin was formalin-fixed and paraffin-embedded, and 5-μm slices of the skin were sectioned with a rotary paraffin microtome (RM2255; Leica) and mounted on positively charged slides. Then slices were steamed for 20 min in 0.01 M sodium citrate buffer (pH 6.0) for antigen retrieval, blocked with 1% normal goat serum (AR0009, Wuhan booster) in PBS buffer for 30 min at room temperature, incubated in primary antibody solution (anti-DCT 1:200, DCT antibodies were kindly gifted from Dr. Ting Chen’s Lab, National Institute of Biological Sciences) at 4°C overnight, washed with PBS buffer for three times, and incubated in secondary antibody solution (1:500). They were mounted with mounting medium with DAPI. Confocal images were acquired with a 10× objective with NA 0.45 on a Zeiss LSM 880 confocal microscope (Nikon).

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.