Axioskop

For the compound microscopy data shown in Fig 4, images were taken using a Zeiss Axioskop with Hamamatsu CCD or ORCA cMOS camera equipped with 63x 1.4NA Plan Apochromat oil immersion objective. Carl Zeiss filter sets 49, 38, and 43HE were used for the visualization of DAPI, Alexa 488, and Alexa 555 respectively. An X-Cite 120Q lamp (Lumen Dynamics) was used as the fluorescence light source. Openlab 5.5.2 (PerkinElmer) and Micromanager [101 (link), 102 (link)] were used as acquisition software. For all other figures, a Leica TCS SP8 confocal microscope driven by LAS software version 3.3.1 or X was used. This laser scanning confocal microscope was equipped with Photomultiplier (PMT) and Hybrid detectors (HyD). For all images, a 63x 1.4NA HC Plan Apochromat oil immersion objective was used with 100–200% zoom for immunostaining, and 300% zoom for single molecule FISH, using the standard scanner with 400Hz scanning speed. For figure preparation, contrast was linearly adjusted in Adobe Photoshop identically across all samples. In some cases, images were merged using the stitching plugin in FIJI/Image J [103 (link)] to generate whole gonad images.

For cross-sectional area (CSA) calculations, 5 μm thick transverse sections were obtained from the mid-belly area of right EDL muscles. After toluidine blue staining, they were randomly selected and photographed using light microscopy (Zeiss Axioskop) with a digital camera (Hamamatsu CCD). Myofiber CSA was measured by tracing the external border of individual myofibers using Image J software. 20 microscopic fields per muscle were analyzed at 20x magnification. Cross-sectional areas were determined for at least 300 muscle fibers per animal. Myofibers exhibiting evidence of tears or processing artifacts were excluded from the analysis.