M4276 clone my 32

Paraffin-embedded biopsies sectioned at 4 μm were deparaffinized with xylene and rehydrated via a series of ethanol washes and distilled water. The rehydrated slide specimens were heated at 95° C in 100 mM citrate buffer (pH 6.0) for 15 min and then allowed to cool for 20 min. Subsequently, the slide specimens were soaked in Super-Sensitive Wash buffer (SS; BioGenex Laboratories, Freemont, CA) for 30 min at room temperature.
A programmable autostainer (BioGenex i6000; BioGenex Laboratories) was used to label sarcolemma and myosin heavy chains. For labeling of myofibers, specimens were blocked with 10% goat serum and were treated with a mixture of mouse anti-type I myosin heavy chain (M8421, clone NOQ7.5.4D; Sigma–Aldrich, St. Louis, MO) and mouse anti-type II myosin heavy chain (M4276, clone MY-32; Sigma–Aldrich) antibodies, for 1 h. Subsequently, the slides were treated for 1 h with a mixture of goat anti-mouse IgG-Alexa Fluor 488 (A11029, Molecular Probes; Life Technologies, Grand Island, NY) and wheat germ agglutinin-Alexa Fluor 647, which label sarcolemma (W32466, Molecular Probes; Life Technologies). Finally, Pro-Long Gold antifade medium with the nuclear label 4′,6-diamidino-2-phenylindole (P36931, Molecular Probes; Life Technologies) was applied to the labeled specimens.