Phalloidin 650

Immunofluorescent assays were performed on cells cultured on glass coverslips. Cells were fixed in 4% formaldehyde, permeabilized with 0.1% Triton X-100 in PBS containing 0.5% Tween20, and blocked with 5% BSA. Primary antibody was a rabbit polyclonal against mitochondrial import receptor subunit TOM20 (#PA5-52843, 1/200; Invitrogen, Merelbeke, Belgium) that had been previously validated by others using shRNAs (Cheng et al., 2009 (link)). Secondary antibodies were goat anti-rabbit Alexa Fluor 488 (#A-11,034, 1/1,000; Invitrogen). For the staining of F-actin, we used phalloidin-650 (1/250; Thermo Fisher). Nuclei were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (1 μg/μl; Sigma-Aldrich). Mitochondrial network analysis was performed using the MiNa toolset on ImageJ following a previously described methodology (Valente et al., 2017 (link)). Cells were counted in 28 different random fields. Images of mitochondrial networks were captured by structured illumination fluorescence microscopy using an ApoTome-equipped AxioImager.z1 microscope (Zeiss, Zaventem, Belgium).

Imaginal discs or adult thorax muscles were dissected in PBS solution (PH 6.8) and fixed in 4% paraformaldehyde in PBS buffer for 30 min. Eye discs or muscles were incubated in diluted primary antiserum, rabbit monoclonal anti-CoIV (1:200) and rat anti-Tom20 (1:200). Anti-rabbit and rat labeled secondary antibodies with Alexa 488, 568, or 647 (1:500) (Invitrogen) were used as secondary antibodies, and Phalloidin 650 (Thermo Scientific) was added to label actin filaments.
For eye section staining, semi-sectioned fly heads were fixed in 4% paraformaldehyde in PBS buffer on ice for 2 hours, and then embedded in LR White resin. Thin sections were prepared at a depth of ~30 μm and incubated with mouse monoclonal anti-Rh1 (1:200) and Rat anti-TRP (1:200) as primary antibodies and Alexa 647 or 750 labeled secondary antibodies (Invitrogen). Samples were examined and images were recorded using a Nikon confocal microscope. The acquired images were processed with Photoshop software.