Rabbit anti laminin

Immunofluorescent stainings for muscle sections and myotubes and immunoblottings were performed with the following antibodies: Rabbit anti‐Msi2 (Millipore, 04‐1069), Mouse anti‐Msi2 (Abnova, H00124540‐M11), Rabbit anti‐HuR (Cell Signaling Technology, 12582), Mouse anti‐HuR (Santa Cruz, sc‐5261), Mouse anti‐Flag tag (Abmart, ab1162), Rabbit anti‐Myc tag (Proteintech, 16286‐1‐AP), Rabbit anti‐Laminin (Abcam, ab11575), Mouse anti‐MyHC (Upstate, 05‐715), Mouse anti‐GAPDH (Protein tech, 60004), Rabbit IgG (Santa Cruz, sc‐2027), Mouse IgG (Santa Cruz, sc‐2025), Rabbit anti c‐Myc agarose beads (Sigma, A7470) and Mouse anti‐Flag agarose beads (Sigma, A2220).

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).

Adult female flies were dissected in phosphate-buffered saline (PBS). All whole digestive tract was removed and fixed in PBS and 4% electron microscopic-grade paraformaldehyde (Polysciences, Warrington, PA, USA) for 35 min. Samples were rinsed 3 times with PBS, 4% bovine serum albumin (BSA), and 0.1% Triton X-100 (PBT-BSA) and incubated with the primary antibody overnight at 4 °C and with the secondary antibody for 2 h at room temperature. Finally, the samples were rinsed 3 times with PBT-BSA and mounted in DAPI-containing Vectashield (Vector Laboratories, Burlingame, CA, USA). All the steps were performed without mechanical agitation. Primary antibodies were goat anti-GFP (1:500; Abcam, Cambridge, UK, ab6673) and rabbit anti-Laminin (1:500, Abcam, Cambridge, UK, ab47651). Secondary antibodies were from Invitrogen (Carlsbad, CA, USA). Phalloidin (Sigma-Aldrich, Merck Life Science UK Limited, Dorset, UK; P1951) was used at 5 µg/mL. Confocal image were acquired with a Zeiss LSM 880. Images were analysed with the Fiji software (National Institutes of Health (NIH) Bethesda, MD) and assembled into figures using Fiji version 1.0, Adobe Photoshop CC 2017 software (Adobe, San Jose, CA, USA), and Microsoft Powerpoint.

Beagles were sacrificed after 12 weeks, and the mandibles were harvested and fixed with 4% paraformaldehyde. Cone-beam computed tomography (CBCT, Vatech, South Korea) was used to observe the harvested samples. All samples were then demineralized for 1 month, embedded, and sectioned into 5-μm-thick sections for histological analyses according to the manufacturer’s recommended protocols. As mentioned earlier, the prepared sections (5 μm) were also stained with H&E stain. For immunohistochemistry analysis, the primary antibodies used in this study were as follows: rabbit anti-DSPP (1:100 dilution; Abcam, MA, United States), rabbit anti-collagen type I (Col-1, 1:200 dilution, Bioss, Inc., Woburn, MA, United States), rabbit anti-laminin (1:50 dilution, Abcam, MA, United States), rabbit anti-DMP-1 (1:50 dilution, Invitrogen, Eugene, OR, United States).