fast twitch myhc type iix  (Developmental Studies Hybridoma Bank)

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 a primary antibody against laminin (ab7463, Rabbit, 1:100) to label the myofiber boundaries, together with a cocktail of mouse monoclonal antibodies against MyHC type I (BA-D5c, MIgG2b. 1:100), MyHC type IIA (SC-71c, MIgG1, 1:100), and MyHC type IIB (BF-F3c, MIgM, 1:100). Primary antibody staining was visualized using Alexa Fluor-conjugated secondary antibodies (1:500) including Goat Anti-Rabbit Alexa Fluor 350 (to detect laminin), Goat Anti-Mouse IgG1 Alexa Fluor 488 (to detect SC-71), Goat Anti-Mouse IgG2b Alexa Fluor 555 (to detect BA-D5), and Goat Anti-Mouse IgM Alexa Fluor 647 (to detect BF-F3). Laminin, BA-D5, SC-71, and BF-F3 staining were pseudo colored white, red, green, and blue respectively. Scale bars 100 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, Bioprocessing

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 a primary antibody against laminin (ab7463, Rabbit, 1:100) to label the myofiber boundaries, together with a cocktail of mouse monoclonal primary antibodies against MyHC type I (BA-D5, MIgG2b), MyHC type IIX (6H1, MIgM), and all MyHC isoforms except type IIX (BF-35, MIgG1). Primary antibody binding was visualized with Alexa Fluor-conjugated secondary antibodies (1:500) including Goat Anti-Rabbit Alexa Fluor 350 (to detect laminin), Goat Anti-Mouse IgG1 Alexa Fluor 488 (to detect BF-35), Goat Anti-Mouse IgG2b Alexa Fluor 555 (to detect BA-D5), and Goat Anti-Mouse IgM Alexa Fluor 647 (to detect 6H1). Laminin, BA-D5, BF-35, and 6H1 staining were pseudo colored white, red, green, and blue, respectively. Scale bars are 100 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, Binding Assay

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: Serial muscle cross-sections (10 µm) of bovine longissimus dorsi muscle biopsy samples were stained with a primary antibody against laminin (ab7463, Rabbit, 1:100) to label the myofiber boundaries together with mouse monoclonal primary antibodies against MyHC type I (BA-D5c, MIgG2b, 1:100), MyHC type IIX (6H1s, MIgM, 1:10), and all MyHC isoforms except type IIX (BF-35c, MIgG1, 1:100), applied either individually or in combination as a cocktail. Primary antibody staining was visualized with a cocktail of Alexa Fluor-conjugated secondary antibodies (1:500) including Goat Anti-Rabbit 350 (to detect laminin), Goat Anti-Mouse IgG2b Alexa Fluor 555 (to detect BA-D5), Goat Anti-Mouse IgM Alexa Fluor 647 (to detect 6H1), and Goat Anti-Mouse IgG1 Alexa Fluor 488 (to detect BF-35). Laminin, BA-D5, BF-35, and 6H1 were pseudo colored white, red, green, and blue, respectively. Scale bars are 100 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: A: Cross-sections (10 µm) of bovine longissimus dorsi muscle biopsy samples were stained with a primary antibody against laminin (ab7463, Rabbit, 1:100) to label the myofiber boundaries in combination with a mouse monoclonal primary antibody against MyHC type I (BA-D5c, MIgG2b, 1:100). Primary antibody binding was visualized using Alexa Fluor-conjugated secondary antibodies (1:500) including Goat Anti-Rabbit Alexa Fluor 647 (to detect laminin) and Goat Anti-Mouse IgG2b Alexa Fluor 555 (to detect BA-D5). Laminin and BA-D5 staining was pseudo colored white and red, respectively. Stitched panoramic images of entire muscle biopsy cross-sections were obtained using an automated fluorescent microscope (Echo Revolution) and analyzed using the MuscleJ 1.0.2 plugin for FIJI/ImageJ with minor custom modification. Segmented myofiber regions of interest (ROIs) were overlaid on the original images and corresponding cartography maps of type I fibers (red) versus non-type I (e.g., type II) fibers (gray) are shown. Scale bars are 400 µm on stitched mosaic images and 100 µm on representative fields of view. B: Comparison of total myofiber count data between manual image analysis and MuscleJ. C: Comparison of the percentage (%) composition of type I (BA-D5 pos ) fibers between manual image analysis and MuscleJ at different sensitivity thresholds for type I fiber identification. D: Comparison of the percentage (%) composition of type II myofibers (BA-D5 neg ) fibers between manual image analysis and MuscleJ at different sensitivity thresholds for type I fiber identification. B-D: Data is presented as the mean ± SEM of muscle biopsy samples from n=13 animals. ** and **** denote p<0.01 and p<0.0001 between the indicated groups, respectively.

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, Modification, Comparison

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: Bovine longissimus dorsi muscle biopsy cross-sections were stained with a primary antibody against laminin (ab7463, Rabbit, 1:100) to label the myofiber boundaries together with a cocktail of mouse monoclonal primary antibodies against MyHC type I (BA-D5c, MIgG2b, 1:100), all but IIX (BF-35c, MIgG1, 1:100), and type IIX (6H1s, IgM, 1:10). Primary antibody binding was detected using Alexa Fluor-conjugated secondary antibodies (1:500) including Goat Anti-Rabbit Alexa Fluor 350 (to detect laminin), Goat Anti-Mouse IgG2b Alexa Fluor 555 (to detect BA-D5), Goat Anti-Mouse IgG1 Alexa Fluor 488 (to detect BF-35), and Goat Anti-Mouse IgM Alexa Fluor 647 (to detect 6H1). Laminin, BA-D5, SC-71, and 6H1 staining was pseudo colored white, red, green, and blue, respectively. 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 with custom modifications. Original images and fiber type cartography maps of type I (red), type IIA (green), and type IIX (gray) 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: 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: Bovine longissimus dorsi muscle cross-section (10 µm) were stained with a primary antibody against laminin (ab7463, Rabbit, 1:100) to label the myofiber boundaries together with a cocktail of mouse monoclonal antibodies against MyHC type I (BA-D5c, MIgG2b, 1:100), MyHC type IIX (6H1s, MIgM, 1:10), and all MyHC types except IIX (BF-35c, MIgG1, 1:100). Primary antibody staining was visualized with Alexa Fluor-conjugated secondary antibodies (1:500) including Goat Anti-Rabbit Alexa Fluor 350 (to detect laminin), Goat Anti-Mouse IgG2b Alexa Fluor 555 (to detect BA-D5), Goat Anti-Mouse IgG1 Alexa Fluor 488 (to detect BF-35), and Goat Anti-Mouse IgM Alexa Fluor 647 (to detect 6H1). Laminin, BA-D5, BF-35, and 6H1 were pseudo colored white, red, green, and blue, respectively. Stitched panoramic images of the entire muscle biopsy cross-section were obtained using an automated fluorescent microscope (Echo Revolution) and analyzed using the MuscleBos plugin for FIJI/ImageJ. An original image and corresponding manually or MuscleBos generated fiber type cartography maps are shown. Scale bars on representative fields of view are 100 µm. B–E: Comparison of manual and MuscleBos analysis of the same bovine muscle cross-sections for total myofiber count ( B ), the percentage of type I myofibers ( C ), the percentage of type IIA myofibers ( D ), and the percentage of type IIX myofibers ( E ). F–H: Comparison of manual and MuscleBos analysis of the same bovine muscle cross-sections for mean myofiber cross-sectional area (CSA) measurements for all fibers irrespective of type ( F ), type I fibers ( G ), type IIA fibers ( H ), and type IIX fibers ( I ). Dot plots represent data from each individual animal (biological replicates) for n=13 cows. NS denotes no significant difference between manual analysis and MuscleBos. * and ** denote p<0.05 and p<0.01 between the indicated groups, respectively.

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, Bioprocessing, Microscopy, Generated, Comparison

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): Muscle biopsy samples were obtained from the longissimus dorsi of high muscle (HM) or low muscle (LM) multiparous dairy cows at 21-days before expected calving (PRE) and at 21-days postpartum (POST). Biopsy cross-sections (10 µm) were stained with a primary antibody against laminin (ab7463, Rabbit, 1:100) to label the myofiber boundaries together with a cocktail of mouse monoclonal antibodies against MyHC type I (BA-D5c, MIgG2b, 1:100), MyHC type IIX (6H1s, MIgM, 1:10), and all MyHC types except IIX (BF-35c, MIgG1, 1:10). Primary antibody staining was visualized with Alexa Fluor conjugated secondary antibodies (1:500) including Goat Anti-Rabbit Alexa Fluor 350 (to detect laminin), Goat Anti-Mouse IgG2b Alexa Fluor 555 (to detect BA-D5), Goat Anti-Mouse IgG1 Alexa Fluor 488 (to detect BF-35), and Goat Anti-Mouse IgM Alexa Fluor 647 (to detect 6H1). Laminin, BA-D5, BF-35, and 6H1 were pseudo colored white, red, green, and blue, respectively. Scale bars are 100 µm. B-J: Stitched panoramic images of the entire muscle biopsy cross-section were obtained using an automated fluorescent microscope (Echo Revolution) and analyzed using the MuscleBos plugin for FIJI/ImageJ. B: The overall fiber type profile of pooled bovine longissimus dorsi muscle biopsy samples showing the percentage composition of type I, type IIA, and type IIX fibers as determined by MuscleBos. C: The mean cross-sectional area (CSA) type I, type IIA, and type IIX myofibers in pooled bovine longissimus dorsi muscle biopsy samples as determined by MuscleBos. D: Comparison of mean myofiber CSA irrespective of fiber type between bovine longissimus dorsi muscle biopsy samples obtained from HM or LM cows at PRE and POST parturition. E-G : Comparison of the mean CSA of type I (E) , type IIA (F) , and type IIX (G) myofibers in bovine longissimus dorsi muscle biopsy samples obtained from HM or LM cows at PRE and POST parturition H-J : Comparison of percentage fiber type composition of type I (H) , type IIA (I) , and type IIX (J) myofibers in bovine longissimus dorsi muscle biopsy samples obtained from HM or LM cows PRE and POST parturition. Values are mean ± SEM with dot plots representing data obtained from each individual animal (biological replicates). Symbols denote *p<0.05, **p<0.01, and ***p<0.001, and ****p<0.0001 between the indicated groups.

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, Bioprocessing, Microscopy, Comparison