Ab8470

Mutations were mapped with structure 4CVH from the RCSB Protein Data Bank (5 ) in YASARA software (10 ) , and the effect of missense mutations was predicted with HOPE (11 ) .
IMMUNOHISTOCHEMISTRY, SDS-PAGE, AND WESTERN BLOTTING Immunohistochemistry, SDS-PAGE, western blotting, and laminin overlay of muscle biopsies and HAP1 or fibroblast glycoproteins enriched with wheat germ agglutinin agarose were performed as described (12, 13 ) . Primary antibodies were desmin (1:100, ab8470, Abcam) and ␤-dystroglycan (1:250, NCL-b-DG, Novacastra). Secondary antibodies were horse radish peroxidaseconjugated polyclonal goat antirabbit or goat antimouse (1:5000, Pierce).

All chemicals used for the generation of fibrin microbeads were purchased from Sigma-Aldrich (St. Louis, MO) and were used without further purification. MSCs isolated from the rat bone marrows were seeded on tissue culture polystyrene (TCPS) flasks (Corning, NY) and cultured in the Minimum Essential Medium Alpha (a-MEM; Lonza, Basel, Switzerland), L- glutamine (Lonza, Basel, Switzerland), penicillin/streptomycin solution (Pen/Strep, Lonza, Basel, Switzerland) and foetal bovine serum (FBS; Lonza, Basel, Switzerland). Trypsin (Sigma-Aldrich, St. Louis, MO) and ethylenediaminetetraacetic acid (EDTA; Sigma-Aldrich, St. Louis, MO) were used to harvest the expanded MSCs. The antibodies used were as follows: anti-MyoD1 antibody (ab16148 AbCam, Cambridge, MA), anti-Desmin antibody (ab8470 AbCam), anti-myogenin (ab1835 AbCam). Whole blood, used as the source of PRP and PPP, was obtained from adult Wistar rats.

FM-R with/without MSCs-transplanted muscle tissues were retrieved at specific time points, fixed in 2.5% GA in phosphate buffer (pH 7.4). Fixed tissue samples were embedded in paraffin and cut into 5-mm sections. After standard preparations, the tissue sections were stained with either haematoxylin and eosin (H&E), or Masson's Trichrome (MTC), then examined under a light microscope (Nikon, Tokyo, Japan). Morphological scoring system of the muscles associated with the stage of repair was implemented on the experimental samples according to Table 1. The regenerative efficiencies of MSC-free FMs, MSCs-containing FMs and the Sham group were compared. The histomorphometric analyses for each experimental group and each time point were performed on nine slides from three different animals for H&E and MTC stainings.
For immunohistochemistry, samples were incubated overnight at 4 C with primary mouse monoclonal antibodies at the respective concentrations: anti-myogenin (1:100; Abcam cat. no. ab1835), anti-Desmin (1:50; Abcam cat. no. ab8470) and anti-MyoD1 (1:100; Abcam cat. no. ab16148). Sections were washed three times with PBS and the secondary antibody was applied for 1 h at room temperature. Sections were washed three times with PBS, and examined under the light microscope (Nikon, Tokyo, Japan).