Immunohistochemistry
SECONDARY
Optimal cutting temperature compound (OCT) embedded muscle samples were transversely orientated on a cutting mount and sectioned at −20 °C (8 μm thickness) on a Cryostat NX70 cryostat (Thermo Scientific, Mass. US), and then mounted on glass slides. Samples were air-dried for 30 min, fixed with 4% paraformaldehyde (15 min), washed (3 × PBS), blocked (60 min), washed (3 × PBS), and incubated (2 h) at room temperature with primary antibodies against laminin (2E8 MIgG2a kappa light chain, 1:100 in PBS, Developmental Studies Hybridoma Bank) and myosin heavy chain type 1 (slow isoform) (A4.84 MIgM0, 1:100, Developmental Studies Hybridoma Bank). After washing (3 × PBS), samples were incubated (1 h) in the dark at room temperature with appropriate secondary antibodies: MHC secondary (Goat anti-mouse IgM mu-chain, 1:200, Alexa 647, abcam), and laminin secondary (Goat anti-mouse IgG2a gamma-chain, 1:250, Alexa 568, Thermo Fisher Scientific). Samples were then washed (3 × PBS), dried, and mounted with cover glasses using Mowiol. All images were captured digitally (LAS X software; Leica Microsystems GmbH) using a Leica DMi8 S widefield fluorescence microscope (Leica Microsystems GmbH) coupled to a Hamamatsu C11440-22C camera (Hamamatsu Photonics, Shizuoka, Japan) at ×20 magnification [Epifluorescence signal was recorded by using excitation filters for laminin (Texas Red, 540–580 nm), and MHC-I (Y5, 620–650 nm)]. Fiber type and mean fiber CSA were obtained using the MuscleJ macro [