Mach1 kit

Muscles were fixed in a solution of acetone, methanol, and water (2:2:1) for 12 h, washed in tap water and dehydrated with ethanol at 70, 96, 100% v/v. After dehydration, the tissue pieces were placed in xylol for 1.5 h and embedded into paraffin. The embedded muscles were sliced into sections (5 μm) that were mounted on glass slides. For immunohistochemical analysis, the serial sections were incubated in an “antigen unmasking solution” (10 mM tri-sodium citrate, 0.05% Tween-20) for 10 min at 75 °C. Then, the MACH1 kit (M1u539 g, Biocare, Concord, CA, USA) was used according to the manufacturer's instructions. The sections were incubated in primary antibodies, including anti-myosin heavy chain-I (MHC-I, A4951, Hybridoma Bank, Iowa, IA, USA), anti-myosin heavy chain-IIa/IIx (MHC-IIa/IIx, A4.74, Hybridoma Bank, Iowa, IA, USA) or anti-myosin heavy chain-IIb (MHC-IIb, BF-F3, Hybridoma Bank, Iowa, IA, USA), in a humidified chamber overnight at 4 °C. The following day, the sections were incubated for 1 h with the secondary antibody and polymers as previously described [34 (link)]. Finally, the slides were cover-slipped, and images were captured with a Leica DM5000 upright microscope (Leica Microsystems, Heidelberg, Germany). By examining serial cross-sections stained for MHC-I and MHC-IIA/X, it was also possible to identify type IIB fibers because of the negative staining.