Automated wide field light microscope

After careful dissection, the SOL and EDL muscles were embedded in optimal cutting temperature (OCT) gel and sectioned at 10 μm with a cryostat (Microtome Plus). The sections underwent blocking for 1 h with M.O.M. mouse IgG Blocking Reagent (Vector Laboratories, MKB‐2213). Sections were then incubated for 1 h with concentrated primary antibodies (BA.D5, SC.71, and BF.F3 all at 1:100 from DSHB). To detect myosin heavy chain I (MHC I), MHC IIa and MHC IIb sections were incubated for 1 h with the following secondary antibodies: Alexa Fluor 647 (1:250; Invitrogen, A21242), Alexa Fluor 488 (1:500; Invitrogen, A21121), and Alexa Fluor 555 (1:500; Invitrogen, A21426). Negative stained fibres were considered to be IIx. After staining, slides were mounted with mounting medium (Vector Laboratories, H‐1000). Slides were imaged with an automated wide‐field light microscope (Nikon Corp.) utilizing a 10× objective lens. The cross‐sectional area of these fibres was then quantified utilizing ImageJ software.

After careful dissection, the SOL and EDL muscles were embedded in optimal cutting temperature (OCT) gel and sectioned at 10 µm with a cryostat (Microtome Plus). The sections underwent blocking for 1 hr with M.O.M. mouse IgG Blocking Reagent (Vector Laboratories, MKB-2213). Sections were then incubated for 1 hour with concentrated primary antibodies (BA.D5, SC.71, BF.F3 all at 1:100 from DSHB). To detect myosin heavy chain I (MHC I), MHC IIa, and MHC IIb sections were incubated for 1 hr with the following secondary antibodies: Alexa Fluor 647 (1:250; Invitrogen, A21242), Alexa Fluor 488 (1:500; Invitrogen, A21121), and Alexa Fluor 555 (1:500; Invitrogen, A21426). Negative stained fibers were considered to be IIx. After staining, slides were mounted with mounting medium (Vector Laboratories, H-1000). Slides were imaged with an automated wide-field light microscope (Nikon Corp.) utilizing a 10x objective lens. The cross-sectional area of these fibers was then quantified utilizing ImageJ software.