Anti-Dystrophin antibody (ab15277)

Muscle stem cells (from normal mouse) were injected into the gastric muscle of an MDX mouse. Dystrophin staining: primary antibody ab15277 and secondary antibody is donkey anti-rabbit Alexa 594.

This image was kindly supplied as part of the review submitted by Jessica Tebbets.

Immunofluorescence staining of dystrophin in W9, W987, and ESC. Myosin heavy chain (MHC) identified muscle cells after differentiation. DAPI was used to stain nuclei.

Seventy-two hours before engraftment, 8 week-old mdx/SCID mice received 14 Gy of irradiation localized to the hind limb muscles. On the day of engraftment, SM/C-2.6-positive myogenic cells were purified by fluorescence-activated cell sorting (FACS), using a BD Aria II FACS machine and the same labeling protocol as described above for FC analysis, resuspended in 30 µl of phosphate buffered saline (PBS), loaded into an insulin syringe (BD), and injected into the left tibialis anterior (TA) muscle of anesthetized mice. 7.5×10⁵ differentiated and sorted W987 cells were injected. Control mice were injected with PBS alone. Three weeks following engraftment, TA muscles were harvested, fixed in 0.5% paraformaldehyde for 4 hours, dehydrated in 20% sucrose overnight and frozen in optimal cutting temperature (OCT) using liquid nitrogen cooled methyl-butane. Tissue blocks imbedded in OCT were cryosectioned and processed for immunocytochemical analysis using rabbit anti-dystrophin. Secondary antibodies used were donkey anti-rabbit conjugated to Alexafluor 594 and donkey anti-rat conjugated to Alexafluor 488 (Life Technologies). Nuclei were visualized using NucBlue Fixed Cell Stain (Life Technologies).

Gene-corrected mdx iPSC W987, non-gene-corrected unexcised mdx iPSC W9 and wild-type ESC controls.

Representative images of longitudinal sections of triceps muscle from 12-week old wild type, FRG1 and FRG1/FHL1 mice co-stained for dystrophin to outline the muscle fiber membrane and DAPI to detect nuclei. Boxed region indicates area shown in high magnification image inset. Scale bars = 100 μm.

For the determination of nuclei number per mm myofiber length, 10 μm longitudinal cryosections from triceps muscle were fixed in 4% paraformaldehyde, washed in PBS and permeabilised/blocked (10% horse serum, 1% BSA and 0.1% triton X-100 in PBS) for 1 hour at room temperature. Sections were stained overnight with rabbit anti-dystrophin (Abcam Cat# ab15277, RRID:AB_301813, 1:400), washed in PBS and incubated with goat anti-rabbit Alexa Fluor 594 and DAPI (1:100) for 1 hour at room temperature. Sections were washed in PBS and coverslips mounted using fluoromount mounting media.

Images of Dystrophin/DAPI stained longitudinal sections were captured using an Olympus BX-51 microscope using dotSlide Software.

Dystrophin quantification in a population of myofibres identified in entire muscle sections performing the double labelling anti-dystrophin ab15277 (red; 1/200 dilution) and anti-spectrin (green; 1/20 dilution).

All the labellings were performed at RT. Muscle sections were incubated with the primary antibody combination (anti-dystrophin ab15277 and anti-spectrin) for 1 hour. After three washes with PBS sections were incubated with Alexa Fluor 488 conjugated anti-mouse IgG (1:100, Thermo Fisher Scientific, Hemel Hempstead, UK) and anti-rabbit biotinylated IgG (1:200; GE Healthcare, Amersham Pl, UK) for 30 minutes. PBS washes were performed and sections were incubated with Alexa Fluor 594 streptavidin conjugate (1:1000, Thermo Fisher Scientific, Hemel Hempstead, UK).

Representative images of entire muscle sections stained and acquired by the Axio Scan slide scanner and processed with Definens algorithm derived from a control (a) and from a DMD patient (b).

DMD: Duchenne Muscular Dystrophy.