Immunofluorescence and microscopy

The iliopsoas or TA/EDL were cryosectioned to a depth of 1 mm from the proximal side, then 7 μm sections were cut and mounted on glass slides for histological and fiber type analysis. Sections analyzed were derived from a comparable 300 μm zone of Ilio or TA, respectively, across all samples, accounting for minor variations in muscle dissection and a subset of samples that required re-sectioning. Immunofluorescent staining for myosin isoforms was performed using mouse monoclonal myosin heavy chain antibodies F1.652 (embryonic), BF-35 (all but 2x), BF-F3 (type 2b), SC-71 (type 2a), and BA-D5 (type 1) (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) at 1:20–1:40 dilution. Samples were co-stained for nuclei (DAPI; Life Technologies, Grand Island, NY) and membrane/basement membrane counterstain by perlecan (heparin sulfate proteoglycan; EMD Millipore, Darmstadt, Germany) or αDGct rabbit monoclonal 5–2 [28] or related hybridomas 29–10 or 45–4, not previously reported. Neuromuscular junctions were analyzed using rabbit monoclonal antibody D35E4 against presynaptic marker synaptophysin (Cell Signaling Technology, Danvers, MA) and Alexa 488-coupled bungarotoxin for detection of nicotinic acetylcholine receptors at the postsynaptic endplate (Life Technologies). Muscle sections were blocked in 5% donkey serum in PBS for 30 min, incubated in primary antibody in 5% donkey serum at 4°C overnight, washed 3 x 5 min, incubated in secondary antibody (AlexaFluor A546 anti-mouse IgG1, IgG2b, or IgM and AlexaFluor A488 anti-rabbit or rat IgG; Life Technologies) with or without A488-bungarotoxin at 1:500 in 5% donkey serum for 30 min at room temperature, washed 3 x 5 min and mounted with PermaFluor (ThermoScientific, Waltham, MA). Detection of glycosylated αDG by indirect immunofluorescence with IIH6 antibody has been described previously [15]. Tissues were viewed by 20X objective on an inverted epifluorescent microscope (Olympus, Center Valley, PA) and images were captured using a DP-72 camera and CellSens software (Olympus).

For image analyses, a series of overlapping images crossing the entire muscle section were taken and compiled into a section map in Photoshop (Adobe). Compiled maps were analyzed by blinded observers in Image Pro Express (Media Cybernetics, Rockville, MD) for fiber counts and whole section area; a subset (all knockouts at 14 d post-CTX) was also analyzed for single fiber area of embryonic myosin heavy chain-positive (eMHC) fibers. Whole section areas from compiled maps were compared for serial sections of the same muscle and the most representative section map for each muscle was selected for total fiber counting. Mean Ilio section areas were: Myf5/Fktn 2 wko LC 0.72 ± 0.21 mm²; KO 0.56 ± 0.18 mm²; 4 wko LC 1.08 ± 0.20 mm²; KO 0.96 ± 0.13 mm²; 8 wko LC 1.73 ± 0.35 mm²; KO 1.21 ± 0.29 mm²; two-way ANOVA age, p < 0.05. Mean TA/EDL section areas were: Myf5/Fktn 2 wko LC 1.20 ± 0.05 mm²; KO 1.89 ± 0.29 mm²; 4 wko LC 2.72 ± 0.39 mm²; KO 1.95 ± 0.12 mm²; 8 wko LC 5.13 ± 0.64 mm²; KO 3.25 ± 0.46 mm²; two-way ANOVA genotype*age, p < 0.05; age, p < 0.0001. Any serial section that deviated by ≥ 15% from the counted map was also counted separately for total fibers. Total fiber counts ranged from 246 to 1716 in the iliopsoas and from 920 to 3795 in the TA of 2, 4, and 8 wko Myf5/Fktn mice; total fibers ranged from 1157 to 7483 in TAs of Myf5/Fktn or Tam/Fktn mice enrolled in the CTX study. Positive fibers (centrally nucleated [CN], eMHC) were counted by manual tag and divided by the number of total fibers in the section map x 100. Proportions of mature fiber types (MHC type 1, 2a, or 2b) were analyzed as the percentage of a given fiber type per total mature fibers (total fibers within a muscle section less eMHC expressing fibers) for each sample. Proportions of MHC type 2x fibers were determined as the percentage of fibers not stained by the MHC “all but 2X” antibody, which detects all MHC isoforms except type 2x, per total mature fibers for each sample.