Transverse sections of tissue samples were sectioned (8 μm thick) and co-stained with rabbit-anti-dystrophin (Abcam) and rat anti-laminin (Sigma) as previously described30 (link). Dystrophin was quantified relative to laminin co-stain using ImagePro software (MediaCybernetics) and normalised to C57CL/10. Briefly, 4 images of dystrophin and the corresponding laminin fields were taken for each section. Following the Arechavala-Gomeza approach54 (link), 10 regions of interest were randomly placed on the laminin image which was overlaid on the corresponding dystrophin image to attain the minimum and maximum fluorescence intensity (Image-Pro, Media Cybernetics, Inc.). Data was normalised relative to C57BL/10. The percentage recovery score was calculated using the following equation: (dystrophin recovery of treated mdx mice-dystrophin recovery of untreated mdx mice)/(dystrophin recovery of C57BL/10 mice-dystrophin recovery of untreated mdx mice); as described on the TREAT-NMD website (http://www.treat-nmd.eu/downloads/file/sops/dmd/MDX/DMD_M.1.1_001.pdf ).
Rabbit anti dystrophin
Manufactured by Abcam
Sourced in United Kingdom
Rabbit-anti-dystrophin is a primary antibody product that recognizes the dystrophin protein. Dystrophin is a key structural component of muscle cells. This antibody can be used to detect and study the dystrophin protein in various applications, such as western blotting, immunohistochemistry, and immunocytochemistry.
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Lab products found in correlation
Rat anti laminin, by Merck Group (2 mentions)
Rabbit anti laminin, by Abcam (2 mentions)
Image pro, by Media Cybernetics (1 mentions)
Rat anti f4 80, by Bio-Rad (1 mentions)
Mouse anti myod, by Agilent Technologies (1 mentions)
Sea block, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Mouse on mouse kit, by Vector Laboratories (1 mentions)
Alexa fluor 488 conjugated goat anti chicken igg, by Thermo Fisher Scientific (1 mentions)
Eclipse ni u, by Nikon (1 mentions)
Chicken anti gfp, by Abcam (1 mentions)
Leitz dm rbe fluorescent microscope, by Leica (1 mentions)
Alexa 594 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Axiovision rel 4, by Zeiss (1 mentions)
Image pro software, by Media Cybernetics (1 mentions)
Ibind flex western device, by Thermo Fisher Scientific (1 mentions)
Mouse anti actinin, by Merck Group (1 mentions)
Ripa buffer, by Roche (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
6 protocols using rabbit anti dystrophin
1
Dystrophin Quantification in Muscle Samples
2
Immunofluorescence Analysis of Muscle Markers
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Sections were fixed with ice-cold 2% paraformaldehyde for 5 min at 4°C, blocked (PBS with 10% NGS and 1.5% BSA), and incubated with primary antibody overnight at 4°C (rabbit anti-dystrophin, Abcam, 1:200 dilution; rabbit anti-laminin, Abcam, 1:80 dilution; mouse anti-Myosin3, DSHB, used neat; mouse anti-Pax-7, DSHB, neat; mouse anti-MyoD, DAKO, 1:500; chicken anti-laminin, Abcam, 1:250; mouse anti-caveolin 1, Abcam, 1:100; and rat anti-F4/80, AbD Serotec, 1:200). The appropriate Alexa secondary antibody (Invitrogen, Carlsbad, CA) was used for detection of each primary antibody, and nuclei were counterstained using 4,6-diamidino-2-phenylindole (DAPI, 1:10000).
3
Immunofluorescence Staining of Muscle Samples
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Immunofluorescence and histological staining were performed as previously described 55 . Gastrocnemius (GC) muscle samples were flash‐frozen in liquid nitrogen, 2‐methylbutane solution. Frozen sections (10‐μm) were fixed with 4% paraformaldehyde for 10 minutes. Sections were washed with phosphate‐buffered saline and permeabilized with 0.3% Triton X‐100 for 5 minutes, and then blocked with Seablock (Thermo Fisher Scientific, Waltham, MA) for 1 hour at room temperature. Cells or frozen sections were incubated at 4°C overnight with a primary antibody as follows: rabbit antidystrophin (1:200, Abcam, Cambridge, MA), antidystrophin‐PE (1:300, SC Biotechnology, Santa Cruz, CA), rabbit anti‐laminin 1 antibody (1:100, Abcam, Cambridge, MA), mouse anti‐Pax7 (1:100, DSHB, Iowa), or mouse antiembryonic myosin heavy chain (1:50, eMyHC, DSHB, IA). A Mouse on Mouse kit (Vector Laboratories, Burlingame, CA) was used per conditions according to the manufacturer's protocol. Secondary antibodies used were donkey anti‐rabbit or anti‐mouse Alexa 488‐ and Alexa 594‐conjugated IgG (Invitrogen and Jackson ImmunoResearch, West Grove, PA, respectively) per the manufacturer's instructions. Nuclei were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI, 1:1,000, Invitrogen). Image acquisition was performed with a Leica Microsystems Inc. (Buffalo Grove, IL) camera at ×4–40 magnification.
4
Immunohistochemical Analysis of Dystrophin, Laminin, and GFP
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Tissue sections were re-warmed for 30 min at RT and blocked with 5% BSA for 1 h at RT, followed by incubation overnight at 4 °C with one or two of the following primary antibodies: rabbit anti-dystrophin (1:200; Abcam), rabbit anti-laminin (1:200; Abcam), and chicken anti-GFP (1:1000; Abcam). After washing, the tissue sections were incubated for 1 h at RT with one or two of secondary antibodies: Alexa Fluor 350-conjugated goat anti-rabbit IgG (1:1000; GeneCopoeia, Rockville, MD, USA) and Alexa Fluor 488-conjugated goat anti-chicken IgG (1:1000; Invitrogen). Immunoreactivity was visualised using a fluorescence microscope (ECLIPSE Ni-U; Nikon, Tokyo, Japan).
5
Quantifying Dystrophin Restoration in Tissue
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Transverse sections of tissue samples were sectioned (8 μm thick) and co-stained with rabbit-anti-dystrophin (Abcam) and rat anti-laminin (Sigma) and probed using goat-anti-rabbit IgG Alexa 594 and goat-anti-rat IgG 488 secondary antibodies respectively (Invitrogen). Dystrophin restoration was quantified by acquiring 4 representative images of dystrophin staining and the correlating laminin field for each section, using a Leitz DM RBE fluorescent microscope (Leica) and Axiovision Rel 4.7 Software (Zeiss). Using the ImagePro software (MediaCybernetics), 10 regions of interest were randomly allocated on the laminin image which was overlaid on the corresponding dystrophin image to attain the minimum and maximum fluorescence intensity for each treatment. Data were extrapolated and normalised to C57BL/10 as previously described37 (link).
6
Protein Extraction and Western Blot Analysis
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Total protein was extracted from the patient’s cardiac and skeletal muscle specimens using RIPA buffer containing protease inhibitors (Roche, Indianapolis, IN, USA). The lysates were sonicated on ice and centrifuged at 20,000 × rpm for 30 min at 4 °C. The supernatant was collected, and protein concentrations were determined employing a BCA protein assay kit (Thermo Fisher Scientific). After mixing with NuPAGE LDS Sample Buffer (Thermo Fisher Scientific), cell lysates were denatured at 70 °C for 10 min, electrophoresed utilizing a NuPAGE Novex Tris-acetate gel 3–8% (Invitrogen) at 150 V for 70 min, and then transferred to PVDF membranes. The membranes were incubated with primary antibodies, followed by incubation with a secondary antibody using the iBind Flex Western Device (Thermo Fisher Scientific). The following primary antibodies were utilized: rabbit antidystrophin (1:500, Abcam, UK; ab15277), mouse antiactinin (1:1000, Sigma-Aldrich, UK; A7811) and antitubulin antibody (1:1000, Sigma-Aldrich, UK; T6199); Histofine Simple Stain MAX-PO (1:100, NICHIREI BIOSCIENCE INC., Tokyo, Japan; 424151) was used as a secondary antibody. Proteins were detected utilizing the ECL Prime Western Blotting Detection Reagent (GE Healthcare, UK; RPN2232), and a ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA). The data were analyzed with Image Lab 6.0 (Bio-Rad).
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