Immunofluorescence Imaging and Morphometry of Skeletal Muscle

Soleus muscles were embedded in paraffin blocks, sectioned into 10 μm slices and triple immunofluorescence staining was performed using and anti‐slow skeletal myosin heavy chain antibody (ab11083, Abcam, Cambridge, MA, USA), anti‐fast skeletal myosin heavy chain antibody (ab91506, Abcam, Cambridge, MA, USA), and anti‐wheat germ agglutinin (W6748, Thermofisher Scientific, Waltham, MA, USA) as previously described (Mortreux et al., 2021 (link)). Stained slides were subsequently imaged at 20× using a Zeiss Axio Imager M1 epifluorescence microscope, and myofiber cross‐sectional area (CSA) was measured using the muscle morphometry plug‐in (Anthony Sinadinos using Eclipse IDE) and FIJI (FIJI, ImageJ, NIH), with experimenters being blinded during both image acquisition and image analysis. CSA data from normally loaded controls (NL R + 0) and hindlimb suspended controls (HLS R + 0) were obtained from animals whose results were the object of another publication (Mortreux et al., 2021 (link)); however, no data were duplicated.