Fluorescence microscopy

For immunofluorescence experiments in TA muscle/single fiber and SC of mouse we followed a standard protocol [27, 59, 60]: samples were fixed with Paraformaldehyde (PFA) 4% for 10 min and permeabilized with 0.1% TritonX-100 in PBS 5 min, then blocked for 1 h in blocking buffer containing 5% normal goat serum and PBS. All primary antibodies (Supplementary Table S1) were diluted in blocking buffer and incubated overnight at 4 °C. Samples were washed three times with PBS and incubated with fluorophore-conjugate (Alexa-conjugates) secondary antibodies for 1 h at room temperature and nuclei were counterstained with DAPI (1:1000), for nuclei detection. Slides were mounted with Fluoreshield Mounting medium. Single myofibers were obtained from isolated TA muscles after 4% PFA fixation (1 h at room temperature): myofibers were dissected under a stereomicroscope and collected in PBS, then stained following the standard immunofluorescent protocol. Confocal images were acquired on a TCS SP8 System equipped with a DMi8 inverted microscope and a HC PL APO 40×/1.30 Oil CS2 (Leica Microsystems, Wetzlar, Germany) at a resolution of 1024 × 1024 pixels (single stack).

Drosophila thoraxes were collected and immersion-fixed for 2 h in cold 4% paraformaldehyde in 0.1 M Phosphate Buffer (PB) at 4 °C. Samples were then transferred to cold 20% sucrose in PB and stored at 4 °C for at least 24 h. Longitudinal sections of DLM (20 μm) were obtained by a cryostat, mounted onto positive charged slides, and stored at −20 °C until use. For immunostaining detection, sections were washed in PB and then pre-incubated for 1 h at room temperature with 5% BSA and 10% of normal goat serum in PB containing 0.5% Triton X-100. Pre-treated sections were incubated for 48 h at 4 °C with the primary antibodies listed in Supplementary Table S1 in PB containing 0.5% Triton X-100. GFP antibody was used to enhance the signal of fluorescent SC in Zfh1>GFP flies. Following washes in PB, the sections were incubated in the appropriate fluorophore-conjugate (Alexa-conjugates) secondary antibodies in PB overnight at room temperature. Images were acquired by a LSM 710 confocal microscope and a Plan-Apochromat 63×/1.40 Oil DIC M27 or EC Plan-Neofluar 40×/1.30 Oil DIC M27 (Carl Zeiss, Oberkochen, Germany) at a resolution of 1024 × 1024 pixels. The distance between adjacent focal planes (z-stacks) was set at 1 µm. Fluorescent phalloidin (F-actin staining, 1:1000) was used to observe Drosophila muscle structure and TO-PRO™-3 Iodide (1:1000) for nuclei detection.

Incubation in secondary antibodies alone was routinely performed as a negative control. When indicated, images has been analyzed by ImageJ software (National Institutes of Health, Bethesda, MD, USA).