Journal: bioRxiv

Article Title: Rapid High-Throughput Analysis of Bovine Skeletal Muscle Fiber Morphology via Automated Fluorescent Microscopy and MuscleBos software

doi: 10.64898/2025.12.18.695234

Figure Lengend Snippet: A: Cross-sections (10 µm) of bovine longissimus dorsi muscle biopsy samples were stained with a primary antibody against dystrophin [MANDYS1(3B7)s, MIgG2a, 1:10] to label the label the myofiber boundaries, together with a cocktail of mouse monoclonal primary antibodies against MyHC type I (BA-D5c, MIgG2b, 1:100) and MyHC type IIA (SC-71c, MIgG1, 1:100). Primary antibody staining was visualized using Alexa Fluor-conjugated secondary antibodies (1: 500) including Goat Anti-Mouse IgG2a Alexa Fluor 647 (to detect dystrophin), Goat Anti-Mouse IgG1 Alexa Fluor 488 (to detect SC-71), and Goat Anti-Mouse IgG2b Alexa Fluor 555 (to detect BA-D5). Dystrophin, BA-D5, and SC-71 staining was pseudo colored white, red, and green, respectively. B: Serial cross-sections from the same bovine longissimus dorsi muscle biopsy sample shown in panel A were stained with a primary antibody against dystrophin [MANDYS1(3B7)s, MIgG2a, 1:10] to label the label the myofiber boundaries in combination with a cocktail of primary antibodies against MyHC type IIX (6H1s, MIgM, 1:10) and all MyHC isoforms except for type IIX (BF-35c, MIgG1, 1:100). Primary antibody staining was visualized using Alexa Fluor-conjugated secondary antibodies (1:500) including Goat Anti-Mouse IgG2a Alexa Fluor 647 (to detect dystrophin), Goat Anti-Mouse IgG1 Alexa Fluor 488 (to detect BF-35), and Goat Anti-Mouse IgM Alexa Fluor 555 (to detect 6H1). Dystrophin, BF-35, and 6H1 staining were pseudo colored white, red, and green, respectively. Scale bars are 200 µm.

Article Snippet: Primary antibodies tested included those against dystrophin [Developmental Studies Hybridoma Bank (DSHB), MANDYS1(3B7)s, MIgG2a, 1:10], Laminin 1 + 2 (Abcam, ab7463, Rabbit, 1:100), MyHC type I (DSHB, BA-D5c, MIgG2b, 1:100), MyHC type IIA (DSHB, SC-71c, MIgG1, 1:100), MyHC type IIX (DSHB, 6H1s, MIgM, 1:10), and all but type MyHC IIX (DSHB, BF-35c, MIgG1, 1:100).

Techniques: Staining

Journal: bioRxiv

Article Title: Rapid High-Throughput Analysis of Bovine Skeletal Muscle Fiber Morphology via Automated Fluorescent Microscopy and MuscleBos software

doi: 10.64898/2025.12.18.695234

Figure Lengend Snippet: Cross-sections (10 µm) of bovine longissimus dorsi muscle biopsy samples were stained with primary antibodies against either laminin (ab7463, Rabbit 1:100) or dystrophin [MANDYS1(3B7)s, MIgG2a, 1:10] to delineate the myofiber boundaries. Primary antibody binding was visualized using Alexa Fluor-conjugated secondary antibodies (1:500) including either Goat Anti-Rabbit Alexa Fluor 350 (to detect laminin) or Goat Anti-Mouse IgG2a Alexa Fluor 647 (to detect dystrophin). Laminin and dystrophin staining was pseudo colored white. Stitched panoramic images of the entire muscle biopsy cross-section were obtained using an automated fluorescent microscope (Echo Revolution) and analyzed using the MuscleJ plugin for FIJI/ImageJ. Segmented myofiber regions of interest (ROIs) were overlaid on the original images and corresponding cartography maps of segmented muscle fibers colored according to their measured cross-sectional areas (CSA) are shown. Scale bars are 400 µm on stitched mosaic images and 100 µm on representative fields of view.

Article Snippet: Primary antibodies tested included those against dystrophin [Developmental Studies Hybridoma Bank (DSHB), MANDYS1(3B7)s, MIgG2a, 1:10], Laminin 1 + 2 (Abcam, ab7463, Rabbit, 1:100), MyHC type I (DSHB, BA-D5c, MIgG2b, 1:100), MyHC type IIA (DSHB, SC-71c, MIgG1, 1:100), MyHC type IIX (DSHB, 6H1s, MIgM, 1:10), and all but type MyHC IIX (DSHB, BF-35c, MIgG1, 1:100).

Techniques: Staining, Binding Assay, Microscopy

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Directed evolution of liver-detargeted AAV vectors for systemic gene delivery to skeletal muscle and heart

doi: 10.1016/j.omtm.2025.101571

Figure Lengend Snippet: AAV9.AAA.NVG7 has reduced liver biodistribution compared to AAVhu.32 in mice and is comparable in NHP The single vector biodistribution of AAVhu.32 vs. AAV9.AAA.NVG7 with a Spc5.μDys transgene was evaluated by i.v. administration in 10 C57Bl/10ScSn-Dmd mdx /J mice at a dose of 5 × 10 13 vg/kg. Quantification is shown for (A) μDys vg/diploid genome, (B) μDys/TBP mRNA transcripts, and (C) microdystrophin protein expression measured by automated capillary western blot as a percentage of wild-type dystrophin. Gastrocnemius and liver microdystrophin are normalized to wild-type dystrophin levels in gastrocnemius, and heart microdystrophin is normalized to heart wild-type dystrophin levels. Gastrocnemius and heart microdystrophin levels are also normalized to alpha-actinin endogenous control. Two-way ANOVA performed by Šidák’s multiple comparisons test. NHP were dosed with scAAVhu.32 ( n = 3 males) or scAAV9.AAA.NVG7 ( n = 2 males) with a U7.3x-snRNA transgene at a dose of 1 × 10 14 vg/kg. (D) Vector biodistribution and (E) transduction were analyzed at day 92. Skeletal muscle tissues include gastrocnemius, quadriceps, tibialis anterior, biceps, and diaphragm. Heart tissues include atrium, ventricle, and apex. Liver tissues include caudate and left lateral segment. scAAV9 liver vg/diploid genome from Hudry et al. used a dose of 1.1 × 10 14 vg/kg, n = 10 NHP, analyzed at 6 months. Two-way ANOVA performed by Šídák’s multiple comparisons test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. All error bars represent S.D.

Article Snippet: For Jess western blot analysis, the 66–440 kDa fluorescent separation module was used with mouse anti-Dystrophin IgG1 6F11 antibody (Developmental Studies Hybridoma Bank) diluted 1:20 with antibody diluent 2 from the Jess kit (Protein Simple, Bio-Techne).

Techniques: Plasmid Preparation, Expressing, Western Blot, Control, Transduction