This is a knockout-validated antibody summary, based on the publication "Multiplex CRISPR/Cas9-based genome editing for correction of dystrophin mutations that cause Duchenne muscular dystrophy", as cited below [1]. Labome curates formal publications to compile a list of antibodies with unambiguous specificity within Validated Antibody Database (VAD).

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Figure 1. Human Δ48–50 DMD myoblasts were treated with SpCas9, CR1, and CR5 to delete exon 51 and sorted for GFP expression as shown in Figure 2. These sorted cells and untreated control cells were injected into the hind limbs of immunodeficient mice and assessed for human-specific protein expression in muscle fibers after 4 weeks post-transplantation. Cryosections were stained with anti-human spectrin, which is expressed by both uncorrected and corrected myoblasts that have fused into mouse myofibers, or anti-human dystrophin antibodies as indicated. White arrows indicate muscle fibers positive for human dystrophin. Scale bars indicate 100 μm. From [1].

Mouse monoclonal IgG2a

Company: Leica

Antibody: dystrophin

Catalog number: NCL-DYS3

Summary: Mouse monoclonal IgG2a against fusion protein containing amino acids 67 to 713 of human dystrophin (epitope mapping residues 321 and 494). Reacts with human by immunohistochemistry(frozen).

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Figure 2. Additional immunofluorescence images probing human dystrophin expression. Serial sections from regions stained with anti-human spectrin are shown inset in top left. (A-C) Sections from muscles injected with untreated human DMD myoblasts. (D-F) Sections from muscles injected with CR1/5 treated human DMD myoblasts enriched by flow cytometry. White arrows indicate dystrophin positive fibers. From [1].

Immunohistochemistry

Human Δ48–50 DMD myoblasts were treated with SpCas9, CR1, and CR5 to delete exon 51 and transplanted into the hindlimb tibialis anterior. Muscles were incubated in 30% glycerol overnight at 4°C before mounting and freezing in Optimal Cutting Temperature compound. Serial 10 micron sections were obtained by cryosectioning.

5% heat-inactivated fetal bovine serum for 30–60 minutes at room temperature.

1:2 dilution overnight at 4°C.

1:200 dilution goat anti-mouse biotin-XX secondary (Life Technologies #B2763) in blocking buffer for one hour at room temperature.

Signal was amplified with streptavidin-HRP conjugates (1:100, from TSA Kit) in blocking buffer for one hour at room temperature. Finally, cryosections were incubated with tyramide-AlexaFluor488 conjugates (1:100, TSA kit) in manufacturer-provided amplification buffer for 10 minutes at room temperature and visualized with conventional fluorescence microscopy.

Multiplex sgRNA targets were designed to delete exon and restore the dystrophin reading frame in a patient mutated cells. This could be considered an inverse KO.

If the antibody described in this summary is a polyclonal antibody, since polyclonal antibodies are of limited quantity, please inquire the supplier whether any current polyclonal antibody with the same catalog number is exactly the same as the one described in this summary. Sometimes, different bleeds or different animals are used, usually with a different lot number. In such cases, the result in this summary may not apply to the new antibody with the same catalog number.