Immunohistochemical Analysis of Muscle Fiber Types

As described previously [23 (link)], the slides with frozen muscle sections were thawed and air dried for 20 min at room temperature. The sections were incubated in TBST for 5 min and the fixed with 4% paraformaldehyde (PFA) for 10 min. After rinse with TBST, sections were blocked with 3% bovine serum albumin (BSA) and 10% AffiniPure Goat Anti-Mouse IgG + IgM Fab (H+L) (Jackson ImmunoResearch, 115-005-044) TBST solution for 1 h at room temperature. Then sections were incubated with primary antibodies diluted in 3% BSA and 5% donkey serum TBST solution overnight at 4°C. Rabbit anti UCHL1 (abcam 108986) and mouse anti type I and type IIa oxidative MyHC (Developmental Studies Hybridoma Bank, BF-32) were used. Following 3 washes with TBST, sections were incubated with appropriate secondary antibodies conjugated with fluorescent dye Alexa 594 or Alexa 488 diluted in 3% BSA TBST (1:400) for 1 hour at room temperature. Sections were then incubated with Hoechst in TBST (1:15 000) for 5 min, followed by 2 washes with TBST. The sections were mounted with coverslips using Fluomount-G solution (Southern Biotech, 0100–01). Immunostaining images was taken using a confocal microscope (Olympus Fluoview 500, Center Valley, PA).