M8421

To classify the fast-twitch and slow-twitch muscle fibers of the skeletal muscle,
immunohistochemistry was performed using antibodies against the myosin heavy chain that is
specifically expressed in fast-twitch and slow-twitch muscle fibers. All samples were
embedded in paraffin and sliced into 3-µm slices. The prepared slide sections were treated
with 8,000-fold diluted primary antibody (M8421 and M4276; Sigma, St. Louis, MO, USA) at
4°C for 12-hr. Next, EnVision + System HRP Labeled Polymer Anti-Mouse IgG antibody (REF
K4001; Dako, Santa Clara, CA, USA) was added to the sections as a secondary antibody,
which was reacted at room temperature for 30 min. Color was developed using Liquid Dab +
Substrate Chromogen System (Dako), and Mayer’s hematoxylin was used as the counterstain.
The cross-sectional area of ​​the muscle fibers was measured using ImageJ software
(National Institute of Health, Bethesda, MD, USA).

Vastus lateralis samples were obtained from the left leg using the modified Bergström needle procedure [28 ] with aspiration under local anaesthesia with 2% lidocaine solution. Prior to analysis, samples were frozen in liquid nitrogen and stored at -80°C. Serial cross-sections (7 μm) were obtained from frozen samples using an ultratom (Leica Microsystems, Germany). Sections were thaw-mounted on Polysine glass slides, maintained at room temperature (RT) for 15 min and incubated in PBS (3 x 5 min). The sections were then incubated at RT in primary antibodies against slow or fast isoforms of the myosin heavy chains (M8421, 1:5000; M4276; 1:600, respectively; Sigma-Aldrich, USA) for 1 h and incubated in PBS (3 x 5 min). Next, the sections were incubated at RT in secondary antibodies conjugated with FITC (F0257; 1:100; Sigma-Aldrich) for 1 h. The antibodies were removed, and the sections washed in PBS (3 x 5 min), placed in mounting media and covered with a cover slip. Images were captured by fluorescent microscope (Eclipse Ti-U, Nikon, Japan). All analyzed images contained 330 ± 11 fibers. The ratio of the number of stained fibers to the total fiber number was calculated. Fibers stained in serial sections with antibodies against slow and fast isoforms were considered hybrid fibers.

To determine if HIF‐α misexpression influenced the in situ slow and fast twitch paraspinal musculature, paraspinal muscle biopsy was obtained from the concave/convex apex of curvature in the AIS subjects and control subjects as aforementioned. Briefly, paraspinal muscle biopsy was fixed in 4% paraformaldehyde solution for overnight and placed in 70% ethanol at 4°C until parafilm embedding. Parafilm blocks were sectioned at 5 μm thickness. Eventually, paraffin sections were dewaxed in xylene, and rehydrated in a gradient of ethanol and PBS. Heat‐mediated antigen retrieval was conducted at 95°C, 20 min, in citrate buffer, pH 6.0 (S236984‐2, Dako). The muscle samples were subject to immunostaining with anti‐slow twitch muscle related myosin heavy chain type I antibody (M8421, Sigma) 1:200 and anti‐fast twitch muscle related myosin heavy chain type II antibody (M4276, Sigma) 1:200 for 4°C, overnight. Secondary antibody rabbit anti‐mouse IgG Alexa 488 (A‐11059, Thermo Fisher Scientific) was applied at dilution 1:500 for 2 h at room temperature. Finally, the sections were counter‐stained with DAPI (H‐1200, Vector laboratories). Whole slide scanning of images (40× magnification) was captured by Vectra Polaris (Perkinelmer) and images were exported to Image J software (version 1.53r) to measure the percentage of myosin heavy chain using threshold analyses.

The frozen gastrocnemius muscle sections (5 μm) were fixed in −20°C acetone for 10 min. Myofibres were outlined by staining of laminin (L9393, Sigma‐Aldrich, St. Louis, MO), and nuclei were visualized by DAPI. To determine the CSA (in μm²) of normal and denervated muscles (n = 6), Nikon microscope was used for image capture, and approximately 300–400 fibres in muscles of each animal were assessed. Data are represented as CSA distribution frequency (in percentage). The mean CSA of type I and type II fibres in normal (n = 6) and denervated muscles (n = 6) were also calculated. Type I fibres were labelled by monoclonal anti‐Myosin slow (M8421, Sigma‐Aldrich, St. Louis, MO). To probe the distribution of type IIb1 and type IIb2 nuclei, sections were stained with primary antibodies to Smox (sc‐166185, Santa Cruz, Dallas, TX) and Plxdc2 (HPA017268, Sigma‐Aldrich, St. Louis, MO).