Axioobserver inverted fluorescent microscope

Human and mouse snap-frozen muscles were cut at 8 μm using a cryostat (Leica Biosystems, CM1860), mounted on slides, and stored at -80°C. Full details of IHC procedures are provided in Table 2. Briefly, slides were thawed and air-dried at room temperature for 20 min followed by fixation with 4% paraformaldehyde (PFA) for 5 min. Following washing with phosphate-buffered saline (PBS) containing 0.02% Tween (PBST) (3 × 5 min), sections were permeabilized with 0.3% triton x-100 in PBS for 10 min at room temperature before being covered in a blocking solution at room temperature for 1 h. Sections were then incubated in primary antibodies overnight at 4°C. Following washes in PBST (3 × 5 min) sections were incubated in the appropriate fluorescently-conjugated or biotinylated secondary antibodies (then anti-biotin tertiary antibody where appropriate) in the dark at room temperature. Following washing with PBS for 3 × 5 min, sections were then treated with a 0.1% Sudan Black B (SBB) solution in 70% ethanol for 2 min to eliminate lipofuscin autofluorescence and quickly rinsed 5 times with PBST, before the final washes in PBST (3 × 5 min), nuclear labeling with DAPI and mounting. Images of skeletal muscle were captured using a Zeiss Axio-observer inverted fluorescent microscope.