Immunostaining of Frozen Muscle Biopsies

Residual frozen tissue from diagnostic human muscle biopsies were obtained from the University of Iowa Wellstone Muscular Dystrophy Specialized Research Center. Cryosections were prepared on glass slides and maintained at −80 °C. Immunostaining was preformed according to a previously published protocol [90 (link)]. In brief, slides were placed in blocking buffer [PBS2+ (130 mM NaCl, 3 mM Na₂HPO₄, 3 mM NaH₂PO₄, 10 mM EGTA), 0.1% Triton-X100, 0.1% BSA] for 1 h at room temperature. Primary antibodies were diluted in blocking buffer and 40 microliters of primary antibody solution were pipetted onto each sample. Samples were covered with parafilm and incubated in the dark in a sealed humidified chamber for one hour at room temperature. Slides were washed in PBS2+ and stained with appropriate secondary antibodies conjugated to Alexa-488, Rhodamine Red-X, or Texas Red-X (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and under the same conditions as the primary antibody. Slides were washed with PBS2+ and a coverslip mounted with 10 microliters of Vectashield with DAPI (Vector Laboratories, Newark, CA, USA). Slides were imaged on a Leica THUNDER Imager Live Cell & 3D Assay imaging system using a 63X objective (Leica Microsystems, Wetzlar, Germany).