Anti laminin

Oligomycin, antimycin, FCCP, rotenone, AICAR, and Luperox (TBHP) were from Sigma. Antibodies against OXPHOS complexes (ab110413) and PGC-1alpha (ab106814) were from Abcam. Anti-GAPDH was from Cell Signaling (2118), anti-laminin from Dako (Z0097), anti-Tim23 (611223) and anti-RalA from BD (610221). Low glucose differentiation medium for immortalized human myoblasts was DMEM-F12 (US Biological) and for primary human skeletal muscle myoblasts (HSMM, Lonza) was DMEM (Gibco) supplemented with glucose (1 g/l) and 2% HS (Gibco). Galactose medium contained DMEM-F12 supplemented with galactose (1 g/l) and 2% HS.

Muscles were excised, weighted, mounted in Killik embedding medium (Bio-optica), frozen in liquid-nitrogen-cooled isopentane, and stored at -80 °C. Transverse muscle sections (7 μm) were cryosectioned from the middle part of each muscle. Myofiber areas were determined by immunofluorescence with anti-laminin (1:200; Dako). Slices were fixed in 4% PFA for 20 min, washed, permeabilized with 0.2% Triton X-100 in 1% BSA for 15 min, and blocked with 4% BSA for 30 min. One hour of incubation with primary antibodies was followed by 45 min of secondary antibody (Alexa Fluor 488-anti-rabbit; Thermo Fisher Scientific) at RT. DAPI was incubated for 5 min at RT to visualize nuclei. SDH staining was performed to reveal the oxidative fibers within a muscle. Frozen transverse TA muscles were incubated in a working solution (SUCCINIC DEHYDROGENASE Stain Lyophilized, Bio-optica) for 45 minutes at 37°C. The sections were then rinsed in distilled water, fixed in 4% PFA for 10 minutes, placed in 15% ethanol for 10 minutes, and, finally, mounted with aqueous mounting medium. Images of whole muscle sections were acquired with the slide scanner Pannoramic Midi 1.14 (3D Histech) and cross-sectional areas (CSA) of fibers or SDH staining quantified with ImageJ software (v1.49o).

Histological analysis was performed as previously reported (Reano et al., 2017 (link)). Briefly, TA muscles were frozen in liquid nitrogen-cooled isopentane and mounted in Killik embedding medium (Bio-optica). Transverse muscle sections (7 μm) were cryosectioned from the mid-belly of each muscle. Sections were stained with Hematoxylin (Bio-optica)/Eosin (Merck) to reveal general muscle architecture. For immunofluorescence, after fixing in PFA 4% for 10 min, slices were permeabilized with 0.2% Triton X-100 in 1% BSA for 15 min and blocked with 4% BSA for 1 h. The primary antibodies anti-laminin (1:200; Dako, Agilent Technologies) and anti-CD31 (1:100; Space srl) were incubated overnight at 4°C, while the incubation with the secondary antibody (1:450, anti-rabbit, Alexa Fluor^TM Antibodies) was performed at room temperature for 1 h. Finally, the slices were incubated with Hoechst 33342 (Merck) for 15 min. Images were acquired using Axio Lab.A1 (Zeiss) and quantified with ImageJ v1.49o software.

Tissues sections were processed and analyzed as previously described (Dreyer et al. 2006) with modifications based on the methods from (Fry et al. 2014, 2015). Muscle biopsies from TKA patients were frozen in isopentane and stored at −80°C. Of the 13 subjects in the study, tissue for histology was obtained from 10 (eight females, two males; average age = 68.1 years). Sections (7 μm) were cut using a Leica Cryostat (CM1850UV) set to −20°C. Following treatment with acetone for 5 min, the sections were blocked in PBS for one hour and primary antibody was added overnight at 4°C. Samples were incubated in secondary antibodies and sections were imaged on a Leica Epifluorescence microscope (Leica DM4000B). Primary antibodies used included anti‐laminin (DAKO, Carpinteria, CA, USA; rabbit) (1:2000), anti‐myosin heavy chain I (BA‐D5; mouse IgG2b) (1:50), anti‐myosin heavy chain IIa (SC‐71; mouse IgG1) (1:500), and anti‐myosin heavy chain IIx (6H1; mouse IgM) (1:100) (Developmental Studies Hybridoma Bank (DSHB), Ames, IA. Secondary antibodies included Alexa Fluor‐labeled mouse IgG2b (1:500), Alexa Fluor‐labeled mouse IgG1 (1:500), Alexa Fluor‐labeled mouse IgM (1:500), and Cy3‐labeled anti‐rabbit (1:200) (Molecular Probes, Eugene, OR).