2.2. Multicolor Immunofluorescence Staining and Measurement of Cross-Sectional Area (CSA)

The separated SO muscle was placed on the gum tragacanth and immediately frozen in chilled isopentane with liquid nitrogen and stored at −80 °C until analysis. The mid-belly transverse cryosections of SO muscles (5 μm thickness) were made with LEICA CM1950 cryostat (Wetzlar, Germany). Sections were put on poly-L-lysine-coated slide glasses, fixed in ice-cold acetone, and stained using multicolor immunofluorescence antibodies as described previously [21]. Primary antibody reactions were performed using anti-myosin heavy chain (MHC) type I (BA-F8), anti-MHC IIa (SC-71), and anti-MHC IIb (BF-F3) (DSHB, Iowa City, IA, USA); the secondary antibodies used were anti-mouse Alexa Fluor 350 IgG_2b, anti-mouse Alexa Fluor 488 IgG₁, and anti-mouse Alexa Fluor 555 IgM, respectively (Thermo Fisher Scientific, Waltham, MA, USA). Images were acquired using a BIOREVO BZ-X710 microscope (Keyence, Osaka, Japan). For fiber type analysis, all fibers within the entire cross-section were characterized. At least 600 myofiber cross-sectional areas (CSAs) were measured per group.