For immunocytochemical (ICC) staining, skeletal muscles were removed and embedded in paraffin following 24 h fixation with 4% paraformaldehyde. Sections (5 μm thickness) of soleus and tibialis anterior muscles were prepared for CD31 (dilution 1:50) ICC staining as previously described (Son et al., 2017 (link)). For the secondary antibody, anti-rabbit IgG Alexa 555 (dilution 1:200) was used.
For staining specific muscle fiber types, fresh tissues were embedded in OCT compound (Thermo 6502, Thermo Fisher Scientific, Waltham, MA, USA), and sections (10 μm thickness) were utilized for staining using anti-myosin heavy chain (MHC) I, anti-MHC IIa, and anti-MHC IIb mouse monoclonal antibodies as primary antibodies. For the secondary antibodies, goat anti-mouse IgG2b Alexa 488, goat anti-mouse IgG1 Alexa 555, and goat anti-mouse IgM Alexa 350 antibodies were purchased from ThermoFisher Scientific. Images were generated by the EVOS® XL Core Imaging System (Mil Creek, WA, USA). Then, the cross-sectional areas were measured in ImageJ (NIH) according to the previous report (Son et al., 2020 ).
For staining specific muscle fiber types, fresh tissues were embedded in OCT compound (Thermo 6502, Thermo Fisher Scientific, Waltham, MA, USA), and sections (10 μm thickness) were utilized for staining using anti-myosin heavy chain (MHC) I, anti-MHC IIa, and anti-MHC IIb mouse monoclonal antibodies as primary antibodies. For the secondary antibodies, goat anti-mouse IgG2b Alexa 488, goat anti-mouse IgG1 Alexa 555, and goat anti-mouse IgM Alexa 350 antibodies were purchased from ThermoFisher Scientific. Images were generated by the EVOS® XL Core Imaging System (Mil Creek, WA, USA). Then, the cross-sectional areas were measured in ImageJ (NIH) according to the previous report (Son et al., 2020 ).