Quadruple Immunofluorescence Quantification of OXPHOS

Quadruple immunofluorescence was carried out on transverse muscle sections (10 μm) using antibodies detecting subunits of OXPHOS complexes (Supplementary Table S1). Complex I was detected using an antibody against subunit NDUFB823 (link), and Complex IV using an antibody to mtDNA encoded subunit I (COX-I). Mitochondrial mass was quantified using an antibody to porin, an outer mitochondrial membrane voltage-gated ion channel. Laminin, a basement membrane glycoprotein, was used to label the myofibre boundaries (Supplementary Table S1). Briefly, the sections were fixed in cold 4% paraformaldehyde (Sigma) for 3 min and permeabilised in a methanol (Fisher) gradient (10 min 70% methanol, 10 min 95% methanol, 20 min 100% methanol, 10 min 95%.methanol and 10 min 70% methanol). Non-specific protein interactions were blocked with 10% normal goat serum (Sigma) and incubated with the primary antibodies in a humidified chamber at 4 °C overnight (Supplementary Table S1). Following washes in TBST (Sigma), the sections were incubated with the secondary antibodies for 2 h at 4 °C and subsequently with streptavidin conjugated with Alexa 647 (Life Technologies) for 2 h at 4 °C (Supplementary Table S1). The sections were washed and mounted in Prolong Gold (Sigma). No-primary antibody controls, incubated only with anti-laminin antibody, were processed for each muscle sample.