The diaphragm bundle allotted for histology was embedded in Tissue-Tek OCT freezing medium, frozen in liquid-nitrogen-cooled isopentane, and stored at − 80 °C. We sliced the diaphragm bundles into 10 μm cross sections at approximately − 20 °C using a cryostat (Leica, CM 3050S model). Sections were incubated in 1:200 wheat germ agglutinin (WGA) Texas Red (Molecular Probes) for 1 h at room temperature, washed in PBS (3 × 5 min), permeabilized with 0.5% Triton X-100 solution (5 min), washed in PBS (5 min), and incubated in primary antibodies in a humid chamber (90 min). We used primary antibodies for myosin heavy chain (MyHC) type I (A4.840, 1:15; Developmental Studies Hybridoma Bank) and MyHC type IIa (SC-71, 1:50; Developmental Studies Hybridoma Bank). After the primary antibody incubation, sections were washed in PBS (3 × 5 min) and exposed to fluorescently conjugated secondary antibodies (60 min) (Goat × Mouse IgM Alexa 350 and Goat × Mouse IgG Alexa 488, Invitrogen). Sections were then washed in PBS (3×5 min), allowed to dry, and imaged.
We acquired and merged images using an inverted fluorescence microscope (Axio Observer) and Zen Pro software (Carl Zeiss Microscopy). To quantify fiber type distribution and fiber cross-sectional area, we used semi-automatic muscle analysis using segmentation of histology (SMASH) code, run in MATLAB software (Smith and Barton, 2014 (link)).
We acquired and merged images using an inverted fluorescence microscope (Axio Observer) and Zen Pro software (Carl Zeiss Microscopy). To quantify fiber type distribution and fiber cross-sectional area, we used semi-automatic muscle analysis using segmentation of histology (SMASH) code, run in MATLAB software (Smith and Barton, 2014 (link)).