Prolong diamond antifade mounting medium

For immunostaining of mito-QC and mt-Keima, larval epidermis or adult flight muscle were dissected in PBS and fixed in 4% formaldehyde, pH 7.0, 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 RT. Tissues were incubated with 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 RT. The tissues were washed twice in PBS and mounted on slides using Prolong Diamond Antifade mounting medium (Thermo Fischer Scientific).
For mitolysosome analysis of mito-QC, tissues were dissected in PBS and fixed in 4% formaldehyde, pH 7.0, for 30 min, rinsed in PBS, mounted in Prolong Diamond Antifade mounting medium (Thermo Fischer Scientific), and generally imaged the next day. For mitolysosome analysis of mt-Keima, larvae were dissected and mounted in Prolong Diamond Antifade mounting medium (Thermo Fischer Scientific) for immediate live imaging. Tissues were imaged via sequential excitations (458 nm, green; 561 nm, red) being captured at 578- to 638-nm emission range.

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.

Staining for Wfs1 and Drd2 mRNAs was performed using single molecule fluorescent ISH (smFISH). Brains from two C57Bl/6J 8-week-old male mice were rapidly extracted and snap-frozen on dry ice and stored at −80 °C until use. Ventral striatum coronal sections (14 μm) were collected directly onto Superfrost Plus slides (Fisherbrand). RNAscope Fluorescent Multiplex labeling kit (ACDBio Cat No. 320850) was used to perform the smFISH assay according to the manufacturer’s recommendations. Probes used for staining are mm-Wfs1-C2 (ACDBio Cat No. 500871-C2) and mm-Drd2-C3 (ACDBio Cat No. 406501-C3). After incubation with fluorescent-labeled probes, slides were counterstained with DAPI and mounted with ProLong Diamond Antifade mounting medium (Thermo Fisher Scientific, P36961). Fluorescent images were captured using sequential laser scanning confocal microscopy (Leica SP8). Basescope Duplex Assay was used for the simultaneous visualization of Wfs1 mRNA and the selective detection of exon 2 of Drd2 mRNA. BaseScope assay was performed and assisted following guidelines (BaseScope™ Detection Reagent Kit-RED User Manual) provided by the supplier (ACDBio Cat No. 323810). Probes used for staining are BA-Mm-WFS1-4EJ-C2 (ACDBio Cat No. 724201-C2) and BA-Mm-Drd2-4zz-st1 (ACDBio custom made No. 724211). Slides were counterstained with hematoxylin and images were captured using brightfield microscope.