The TA muscle samples were sectioned in a cryostat (Leica CM1850 UV, Wetzlar, Germany). Immunofluorescence on 10-µm sections detected Dystrophin (Santa Cruz, rabbit anti- Dystrophin #sc-15376) and nuclei (by DAPI, 4′,6-diamidino-2-phenylindole). Photomicrographs were acquired on Axio Scope.A1 (Carl Zeiss Microscopy GmbH, Göttingen, Germany). The muscle fibers’ cross-sectional area (CSA) was measured using the ImageJ software (v. 1.45s, National Institutes of Health, Bethesda, MD, USA). Around 800–1200 fibers were analyzed per group. The overall integrity and architecture of the tissue were evaluated by hematoxylin-eosin stains.
Dystrophin
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Dystrophin is a protein that plays a crucial role in maintaining the structural integrity of muscle fibers. It serves as a key structural component within the muscle cells, providing a crucial link between the internal cytoskeleton and the extracellular matrix. Dystrophin helps to transmit the mechanical force generated by muscle contractions, ensuring the proper functioning and stability of muscle tissues.
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Lab products found in correlation
Cm1850 uv, by Leica (1 mentions)
M o m kit, by Vector Laboratories (1 mentions)
Collagen 1, by Merck Group (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Fluoromount, by Agilent Technologies (1 mentions)
Diaphot inverted microscope, by Nikon (1 mentions)
P smad2, by Abcam (1 mentions)
F4 80, by Abcam (1 mentions)
Pvdf membrane, by Cytiva (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Calpastatin, by Cell Signaling Technology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Calpain 1, by Cell Signaling Technology (1 mentions)
Anti tubulin, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Cm1100, by Leica (1 mentions)
Fluorescent mounting medium, by Agilent Technologies (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
6 protocols using dystrophin
1
Muscle Fiber Analysis via Immunostaining
2
Immunofluorescence Analysis of Extracellular Matrix
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For immunofluorescence, 7-μm cryosections were fixed in 4% paraformaldehyde, blocked for one hour in 10% goat serum in PBS and incubated for one hour at room temperature with specific antibodies against fibronectin (Sigma, St. Louis, MO, USA), collagen I (Chemicon, Temecula, CA, USA), F4/80 (Abcam, Cambridge, MA, USA), p-Smad-2 (Abcam, Cambridge, MA, USA), and dystrophin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). FITC-conjugated goat anti-rabbit IgG and rabbit anti-mouse IgG (Invitrogen, Carlsbad, CA, USA) were used as secondary antibodies. For monoclonal anti-mouse antibodies, all incubations were performed with mouse IgG-blocking solution from the MOM kit (Vector Lab, Burlingame, CA, USA) diluted in 0.01% Triton X-100/PBS. For nuclear staining, sections were incubated with 1 μg/ml Hoechst 33258 in PBS for ten minutes. After rinsing, the coverslips were mounted using Fluoromount (Dako, Carpinteria, CA, USA) and observed under a Nikon Diaphot inverted microscope equipped for epifluorescence [26 (link)].
3
Western Blotting of Cardiac Proteins
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The hearts were collected as described before (n = 5-8/group), the left ventricle walls isolated and homogenized in RIPA buffer with protease inhibitor cocktail (Sigma-Aldrich, Inc., St. Louis, MO, USA). Fifty μg protein/well were resolved on a 5%-16% SDS-Page gels and transferred to PVDF membrane (Amersham Pharmacia Biotech, Amersham, UK). The membranes were blocked with 5% albumin for 2 h and incubated overnight at 4°C with the primary antibodies to dystrophin (1:50, Santa Cruz Biotechnology, Santa Cruz, CA, USA), calpain-1 (1:500, Cell Signaling, Danvers, MA, USA), calpastatin (1:500, Cell Signaling) and alpha-fodrin (1:500, Cell Signaling). After that, they were incubated with HRP-conjugated secondary antibodies for an additional 1 h at room temperature. Equal protein loading of the samples was verified by staining anti-tubulin (Santa Cruz Biotechnology), anti-GAPDH (Cell Signaling, Danvers, MA, USA) or β-actin (1:5000, Santa Cruz Biotechnology). The membranes were developed using ECL (Millipore, Billerica, MA, USA). Gel documentation and signal quantification were made using the Bio-Image Analysis of Molecular Imager ChemiDoc XRS System (Bio-Rad, Richmond, CA, USA).
4
Immunofluorescent Muscle Fiber Typing
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The muscle tissue was cross-sectioned at −22 °C using a cryo-microtome (Leica CM1100, Leica Microsystem Nussloch GmbH, Nussloch, Germany). The muscle tissue sections were mounted on microscope slides and placed in a freezer (−20 °C) overnight. The next day, the slides were thawed, permeabilised in 0.01 M phosphate-buffered saline (PBS) containing 0.25% Triton X-100 (15 min) and washed with PBS (3 × 5 min). The myofibres were labelled with MHC II (myosin heavy chain II, 1:250; A4.74, mouse monoclonal antibody, Developmental Studies Hybridoma Bank, Iowa City, IA, USA) primary antibody for 1 h, after which positive fibres were observed with an alexa fluor 488 secondary antibody conjugate (1:250, goat anti-mouse, Invitrogen, Eugene, OR, USA) to discern the type II muscle fibres. To observe the sarcolemmas, myofibres were incubated with dystrophin (1:250, rabbit polyclonal, Santa Cruz Biotechnology, Santa Cruz, CA, USA) primary antibody and alexa fluor 594 (1:250, goat anti-rabbit, Invitrogen, Eugene, OR, USA) secondary antibody conjugate for 1 h at room temperature. All sections were labelled with bisBenzimide H 33342 trihydrochloride (1:200, Hoechst, B2261, Merck, Darmstadt, Germany) to visualise the nuclei. The tissue sections were then rinsed in PBS (3 × 5 min) and mounted with a fluorescent mounting medium (DAKO; GLostrup, Denmark).
5
Muscle Biopsy Analysis of BMD
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An open muscle biopsy was performed on the nondominant biceps brachii under local anesthesia with separate quadrants sampled at baseline and 8 weeks in six patients. The biceps brachii was chosen due to relative preservation of this muscle in BMD vs shoulder girdle, pelvic girdle, and thigh musculature and the lack of deleterious effect of biopsy on lower extremity clinical testing. The western blot methodology is detailed in the Supporting Information online. All specimens were assessed with the technician blinded to the baseline vs treatment status. Antibodies used for quantification of bioenergetic signaling proteins, mitochondrial proteins, structural muscle proteins, indicators of regeneration, and regulators of muscle growth were from the following sources: 5′‐adenosine monophosphate (AMP)‐activated protein kinase (AMPK), LKB1, PGC1α, mitofilin dysferlin, follistatin, myostatin, Myf5, MyoD, myogenin, and MEF2a (all from Abcam, Cambridge, Massachusetts, USA); dystrophin, utrophin, creatine kinase, myosin, and actin α1 (all from Santa Cruz Biotechnology, Santa Cruz, California); and glyceraldehyde 3‐phosphate dehydrogenase (all from Cell Signaling, Danvers, Massachusetts), which was used as loading control. Band intensities were digitally quantified using ImageJ software (National Institutes of Health, Bethesda, Maryland; http://www.nih.gov ).
6
Immunofluorescence Staining of Muscle Markers
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With immunofluorescence staining, the expression of markers was detected using antibodies against desmin (Proteintech), PAX7 (Proteintech), MYHC (Thermo Fisher Scientific), MYOD (Invitrogen), F-actin (Abcam) and dystrophin (Santa Cruz Biotechnology, TX, USA). Briefly, cell samples were washed thrice with PBS and then fixed with 4% paraformaldehyde for 15 min. After washing with PBS, the cells were permeabilized with 0.25% Triton X-100 (Rich Joint, Shanghai, China) for 10 min and incubated in 5% fetal bovine serum at 37°C for 1 h. Afterward, cells were incubated with primary antibodies overnight. After washing, secondary antibody was used to incubate the cells for 1 h at room temperature. The cells were then washed and treated with Hoechst (Beyotime) to stain the cell nuclei. In the end, samples were imaged under a fluorescence microscope (Olympus). Using ImageJ software according to the method described by Menconi et al. [Citation32, Citation33] , quantitative image analysis was performed by analyzing five randomly selected injury fields per sample; fusion index was calculated as the number of nuclei in myotubes divided by the total number of nuclei counted. The average number of nuclei per myotube was determined by dividing the number of nuclei in myotubes by the total number of myotubes.
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