My 32

Excised gastrocnemius and soleus tissues were fixed with 4% paraformaldehyde and embedded in paraffin. Tissue samples were cut at a thickness of 6 µm and the sections were stained with hematoxylin-eosin (HE). The mean CSA of fibers was determined using the Image J software (National Institute of Health). Immunohistochemical staining was performed with a monoclonal antibody against skeletal slow (NOQ7.5.4D, Sigma) and fast myosin (MY-32, Sigma).

Cells were seeded at ∼90% confluence on chamber culture slides. After 24 h they were induced to differentiate for 9 days in DM containing human serum from either stable, exacerbated COPD patients or healthy controls as described above. Differentiated myotubes were fixed with 100% methanol for 10 min at -20 °C and incubated overnight at 4 °C with a primary antibody specific to fast myosin heavy chain protein (MHC) (MY-32, Sigma-Aldrich). Then, cells were labelled with a donkey anti-mouse antibody conjugated with Alexa Fluor 488 (Jackson ImmunoResearch Laboratories Inc) and DAPI was used for nuclei staining. Treated cells were observed using a fluorescence microscope (Olympus BX61) equipped with a digital camera with appropriate filters.

C2C12 cells were fixed with 4% paraformaldehyde solution for 10 minutes and permeabilized with 0.5% TritonX-100 (Nakarai Chemicals) and 0.1% SDS solution for 10 min. Cells were incubated with rabbit anti-Bach1 polyclonal antibody (1:400 dilution) [18 (link)] or anti-myosin heavy chain antibody (MY32, Sigma) diluted with PBS containing 1% BSA for 1 hr at 37°C. Cells were then incubated with secondary antibody conjugated with sheep anti-mouse IgG-FITC (F3008, Sigma) diluted with PBS containing 1% BSA for 30 min at 37°C. Cells were mounted in VECTASHIELD Mounting Medium with DAPI (VECTOR) or Hoechst. Fusion index was calculated as percentage of nuclei within myosin positive cells with at least 3 nuclei versus total nuclei in the fields.

Freshly isolated skeletal muscle was frozen in liquid nitrogen-chilled isopentane. 10μm sections were used for all immunostaining. Myofiber cross-sectional area and fiber composition were determined as previously described [29 (link)]. Primary antibodies were purchased from Developmental Studies Hybridoma Bank (type 1 fiber—A4.84, type 2a fiber—SC-71, type 2b fiber—BF-F3, and type 2d fiber– 6H1) and Sigma (laminin—L9393, fast myosin—MY32). A4.84 was also used for the detection of primary myofibers in E18.5 embryos. Secondary antibodies were purchased from Southern Biotechnology (anti-mouse IgM-FITC– 1021–02, anti-mouse IgG-555–1030–32) and ThermoFisher Scientific (anti-rabbit IgG-Alexa Fluor 350 –A11046). Staining of E18.5 immunosections were modified as follows. Slides were thawed on ice for 10 min and then fixed in 2% PFA at room temperature for 10 min. After washing in PBS, the sections were permeabilized with 0.1% Triton X-100 for 5 min, blocked in 15% horse serum in 0.1% Triton/PBS for 1 hour, and then incubated with primary antibodies in blocking buffer overnight at 4C. Slides were mounted with Prolong Gold Antifade Mountant (ThermoFisher Scientific), and visualized with the Leica DMI6000B inverted microscope and the Zeiss Axio Observer Inverted fluorescent microscopes and color CCD camera.