Transverse 8 μm thick frozen sections were cut from diaphragm muscle strips. Hematoxylin and eosin (H&E; Sigma-Aldrich) staining was used for muscle morphometric analysis97 (link). Digital images of H&E stained muscle were captured at 200 × magnification (DM1000 upright microscope, Leica). All histology image analysis was completed using Image-Pro Plus software (Media Cybernetics). Muscle fibre size is expressed as minimal ferret diameter to control for variation in the orientation of the muscle cross-section.
WGA is an effective tissue marker for fibrosis67 (link), due to the presence of WGA binding sites in the pericellular and interstitial matrices which in dystrophic muscles are enriched with collagen, proteoglycans, and glycosaminoglycans (e.g. hyaluronan)64 (link),66 (link). Diaphragm cross-sections were fixed in 4% PFA and stained with WGA conjugated with Alexa Fluor 594 (Thermo Fisher Scientific; 1:50 dilution in PBS) for 15 min. Nuclei were counter-stained with DAPI. Two non-overlapping images for each cross-section were captured on an Olympus 1X71 Inverted Fluorescence Microscope with an XM10 camera. To determine the percentage area of fibrosis in the diaphragm cross-sections, planimetric analysis of the digital images was completed using Image-Pro Plus software (Media Cybernetics)14 (link),98 (link).
WGA is an effective tissue marker for fibrosis67 (link), due to the presence of WGA binding sites in the pericellular and interstitial matrices which in dystrophic muscles are enriched with collagen, proteoglycans, and glycosaminoglycans (e.g. hyaluronan)64 (link),66 (link). Diaphragm cross-sections were fixed in 4% PFA and stained with WGA conjugated with Alexa Fluor 594 (Thermo Fisher Scientific; 1:50 dilution in PBS) for 15 min. Nuclei were counter-stained with DAPI. Two non-overlapping images for each cross-section were captured on an Olympus 1X71 Inverted Fluorescence Microscope with an XM10 camera. To determine the percentage area of fibrosis in the diaphragm cross-sections, planimetric analysis of the digital images was completed using Image-Pro Plus software (Media Cybernetics)14 (link),98 (link).
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