Sc 71

10 μm frozen sections were incubated with Mouse on Mouse IgG Blocking (Vector Lab) for 1 h at room temperature then washed 2 times in PBS. Sections were then incubated with a mixture of BA-D5 (Myosin Heavy Chain Type I), SC-71 (Myosin Heavy Chain Type IIa), BF-F3 (Myosin Heavy Chain Type IIb) (Developmental Studies Hybridoma Bank) for 45 min at 37 °C, in PBS-1%BSA. Sections were washed 3 times in PBS for 5 minutes at room temperature and then incubated with a mixture of the following secondary antibodies: anti-mouse IgG2b-Alexa405, IgG1-Alexa488, IgM-Alexa594 (ThermoFisher), all made in goat, diluted in PBS-1%BSA, for 30 minutes at 37 °C. Sections were washed for 5 minutes, 3 times with PBS at room temperature and mounted with ProLong Gold (Invitrogen).

Acetone-fixed 10 μm cryosections of gastrocnemius were rinsed in phosphate-buffered saline with 1% Tween 20 (PBS-T). After washing with PBS-T, the tissue sections were blocked with blocking mouse IgG (Vector Laboratories) and 5% normal goat serum (Thermo Fisher Scientific). The sections were incubated with anti-myosin heavy chain (MHC) I (1:250), anti-MHC IIa (1:250), anti-MHC IIb (1:250) (BA-F8, SC-71, BF-F3; Developmental Studies Hybridoma Bank), and anti-laminin (1:500; Sigma Chemical) antibodies overnight at 4 °C. The following day, the sections were rinsed with PBS-T and incubated with the appropriate secondary antibody conjugated to Alexa Fluor 350, 488, 555, and 647 antibodies, respectively (A21140, A21042, A21127, A21244; Thermo Fisher Scientific). Slides were mounted with ProLong Gold Antifade Mountant (P36930; Thermo Fisher Scientific) and glass coverslips. Fluorescent images were captured using a DMi8 inverted microscope (Leica).

All immunohistochemical procedures in the present study were carried out according to our previous study^{7 (link)}. To detect different myofibers, gastrocnemius muscle Sects. (10 µm-thick) were incubated with antibodies specific to myosin heavy chain (MyHC) types I, IIa, and IIb (BA-D5, SC-71, and BF-F3, respectively, University of Iowa Developmental Studies Hybridoma Bank, Iowa City, IA), supplemented with rabbit polyclonal anti-laminin antibody (L9393; Sigma-Aldrich, St. Louis, MO). Unstained myofibers were judged as MyHC IIx expression. Alexa Fluor 405, 488, and 546 secondary antibodies were used to detect MyHC types I, IIa, and IIb, respectively (Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA). Further, laminin (L9393 Sigma-Aldrich, St. Louis, MO, USA) and Pax7 (Developmental Studies Hybridoma Bank, Iowa, IA, USA) were used to detect myofiber borders and satellite cells, respectively. Secondary antibodies coupled to anti-rabbit IgG Cy3 and Cy5-labeled (Jackson Immunoresearch Labs, West Grove, PA, USA) were used to detect laminin and Pax7.

C57BL/6 mice were injected retro-orbitally with 4.5E + 13vg/kg of AAV9-, AAVMYO-, MyoAAV2A-, MyoAAV4A-CMV-luc-IRES-eGFP and tibialis anterior muscles of each injected mouse were collected and 8 μm thick muscle cross-sections were prepared for immunofluorescence studies. After being fixed with 4% paraformaldehyde and permeabilized with 0.02% Triton 1X, transversal TA sections were washed with PBS 1X and blocked 1 h with BSA 3% (bovine serum albumin) in PBS to avoid nonspecific binding. After, BF-F3 and SC-71 (Developmental Studies Hybridoma Bank, Lowa, US) diluted at 1:50 and eGFP (Invitrogen #A-11,122, US) diluted at 1:100 were used as primary antibodies to detect muscle fiber type IIb and transgene vector expression, respectively. Sections were incubated with primary antibodies overnight at 4 °C in a humidified atmosphere. After series of washing with PBS 1X, sections were treated with secondary antibodies IgM DyLight 405 goat anti-mouse (dilution 1:100), IgG1 Cy5 goat anti-mouse (dilution 1:100) or Alexa Fluor 488 donkey anti-rabbit (dilution 1:400) and during 1 h at room temperature. After series of washing, sections were mounted with ProLongTM Gold antifade reagent (Invitrogen #P36934, Massachusetts, US) and air-dried before imaging using a microscope (Zeiss, Germany).

Primary antibodies against SERCA2a (2A7-A1), PLN (2D12), dynamin 2 (PA5-19800) and RyR (MA3-925) were obtained from Pierce Antibodies. The primary antibody for SERCA1a (A52) was a kind gift from Dr David MacLennan (University of Toronto) (Zubrzycka-Gaarn et al., 1984 (link)). The primary antibody directed against SLN was generated by Lampire Biological Laboratories (Fajardo et al., 2013 (link)). Anti-ubiquitin (P4D1) and anti-nitrotyrosine (189542) antibodies were obtained from Cell Signaling Technology and Cayman Chemicals, respectively. The primary antibody against α-actin (A4700) was obtained from Sigma-Aldrich. The primary antibodies against MHCI (BA-F8), MHCIIa (SC-71), MHCIIb (BF-F3), embryonic MHC (BF-F6) (Schiaffino et al., 1989 (link); Lucas et al., 2000 (link)) and dystrophin (3B7) (Nguyen thi et al., 1990 (link)) were obtained from Developmental Studies Hybridoma Bank. Secondary antibodies for western blotting, goat anti-mouse IgG (peroxidase conjugated) and goat anti-rabbit IgG (peroxidase conjugated) were obtained from Santa Cruz Biotechnology. Secondary antibodies for immunofluorescence staining, Alexa Fluor 350 anti-mouse IgG_2b, Alexa Fluor 488 anti-mouse IgG₁ and Alexa Fluor 555 anti-mouse IgM, were obtained from Molecular Probes.