Immunohistochemical Analysis of Muscle Proteins

Immunohistochemistry of Drosophila larval body wall muscles and human muscle biopsy tissues were performed as previously described [9 (link)]. Drosophila larval body wall muscles were stained with affinity purified anti-CncC antibodies (1:100; gift from H. Deng and T. Kerppola) [24 (link)], anti-p62/Ref(2)P (1:3,000, kind gift of G. Juhász) [55 (link)] that was detected with Alexa Fluor 488 goat anti-rabbit (1:400 dilution; Invitrogen). Filamentous actin was detected with Texas Red Phalloidin (1:400, Invitrogen). Human muscle biopsy cryosections were obtained from the Iowa Wellstone Muscular Dystrophy Cooperative Research Center and stained according to published procedures [9 (link)] with human p62/SQSTM1 (1:3,000, Sigma) and Nrf2 (1:300, Santa Cruz Biotech), followed by Alexa Fluor 488 goat anti-rabbit (1:400, Invitrogen). Filamentous actin was detected with Texas Red Phalloidin (1:400, Invitrogen).