Ncl dys2

Immunohistochemical analysis involved the use of immunoperoxidase techniques on frozen sections. MANDYS110 clone 3H10, provided by the Wolfson Centre for Inherited Neuromuscular Disease, NCL-DYSB and NCL-DYS2 (Novocastra Laboratories, Newcastle on Tyne, UK) mouse monoclonal antibodies were used for dystrophin protein detection (1∶50 for each ones). Other primary antibodies used were IVD3₁A9 (1∶50, Developmental Studies Hybridoma Bank, Iowa City, IA), NCL-g-Sarc (1∶10, Novocastra Laboratories) and NCL-DRP2 (1∶60, Novocastra Laboratories) for alpha-sarcoglycan, gamma-sarcoglycan, and utrophin detection respectively, NCL-MHCd (1∶20, Novocastra Laboratories) for developmental myosin heavy chain isoform, anti-complement 5b-9 (1∶250, Calbiochem, Strasbourg, France) and anti-CD3 (1∶100, Dako, Glostrup, Denmark) for lymphocytes. Briefly, transverse cryosections were incubated in PBS with 5% normal goat serum (Dako) for 1 hour at room temperature. They were then incubated with primary antibody in 5% rat serum overnight at 4°C and with biotinylated secondary antibodies (E433, 1∶300, Dako) in PBS with 5% rat serum for 1 hour. Bound antibodies were detected either with streptavidin (P397; Dako) and DAB Liquid Substrate (Dako) for immunoperoxidase. Fiber type was determined using histochemical myosin-ATPase reaction after preincubation at pH 4.2, 4.35, and 10.4 as previously described [22] .

For western blots, a mouse monoclonal anti-dystrophin antibody (NCL-DYS2, Novocastra, Muttenz, Switzerland) was used at a dilution of 1/250. A mouse monoclonal antibody against SAP97 (VAM-PS005F, Enzo life science, Lausen, Switzerland) was used at a dilution of 1/500. Against Na_v1.5, a custom-made rabbit polyclonal antibody (Pineda Antibody Service, Berlin) was used at a dilution of 1/200. A rabbit polyclonal antibody against Cre (69050; Novagen, EMD Millipore-MERK, Schaffhausen, Switzerland) was used at a dilution of 1/500 and a rabbit polyclonal antibody against calnexin (C4731; Sigma-Aldrich, Saint-Louis Missouri, USA) was used at 1/1000^{11 (link),26 (link),27 (link)}.
For immunostainings, rabbit polyclonal antibody against Cre (69050; Novagen, EMD Millipore-MERK, Schaffhausen, Switzerland) was used at a dilution of 1/500 and mouse monoclonal anti-dystrophin (Dys NCL-DYS2, Novocastra, Muttenz, Switzerland) was used at a dilution of 1/250^{11 (link),26 (link),27 (link)}.

In vitro differentiated immortalized Δ52 myoblasts grown on 35 mm u-dishes (Ibidi, Gräfelfing, Germany) were washed with PBS (Sigma-Aldrich, St. Louis, MO, USA), fixed with 4% paraformaldehyde (Sigma-Aldrich) at room temperature for 10 min, and permeabilized with ethanol 75% (Sigma-Aldrich) at room temperature for 1 min. As a blocking solution, 20% fetal bovine serum (Sigma-Aldrich) was used at room temperature for 30 min to reduce secondary antibody background signal. The cells were subsequently incubated overnight at 4 °C with the anti-dystrophin (NCL-DYS2 Novocastra) primary antibody. After incubation, cells were washed with PBS and then incubated with the 594-fluorochrome conjugated secondary antibodies (Thermo Fisher, Waltham, MA, USA) together with DAPI for nucleic acid staining (Ibidi) for 1 hr at room temperature in PBS containing 0.2% Triton X-100. After three successive washings with PBS, dishes were mounted using fluorescent mounting medium (Ibidi) and examined by fluorescence microscopy (DMI6000B, Leica, Milan, Italy).

Frozen sections of 7 micrometer thickness from muscle biopsy specimens were processed for histology, histochemistry, and immunohistochemistry according to standard protocols [48 ].
Regarding the detection of sarcolemmal and sarcolemma-associated proteins, immunohistochemistry was performed by using antibodies targeting different dystrophin epitopes: NCL-DYS1 (clone: DY4/6D3), NCL-DYS2 (clone: DY8/6C5) and NCL-DYS3 (clone: DY10/12B2), corresponding to central/core, C-terminal and N-terminal regions, respectively (Novocastra, Newcastle, UK, primary antibody dilution was 1:20 in all cases). The following antibodies were used targeting sarcoglycan alpha, beta, gamma, and delta: NCL-L-a-SARC, clone AD1/20A6; NCL-L-b-SARC, clone BEATASARC1/5B1; NCL-g-SARC, clone 35DAG/21B5; NCL-d-SARC, clone DELTASARC/12C1; (Novocastra, 1:50, 1:100, 1:100, and 1:50, respectively); merosin: NCL-MEROSIN, clone: MER3/22B2 (Novocastra, 1:100) and spectrin NCL-SPEC1, clone RBC2/3D5 (Novocastra, 1:100). Primary antibodies were incubated on slides for 1 h at room temperature.
As negative control, a muscle biopsy sample from an ALS (Amyotrophic Lateral Sclerosis) patient was used. We have succesfully applied the settings described above to detect dystrophinopathy recently [49 (link)].