Ab28365

Frozen cross sections of the masseter muscle were immunohistochemically double-stained with antibodies against dystrophin (#D8168, Sigma) and CD31 (#ab28365, Abcam, Cambridge, UK). The number of microvessels per masseter myocyte was calculated as described previously [22 (link),36 (link)].

Immunohistochemistry was performed as described previously [16] (link). In brief, 3-µm-thick sections of the aorta, fixed in 4% paraformaldehyde, were deparaffinized with xylene and graded alcohol from 100% to 70% and autoclaved at 121°C for 15 min in 10 mmol/l citrate buffer (pH 6.0). Following this, section slides were immersed in 3% H₂O₂ for 20 min to block endogenous peroxidase activity, followed by 10 min in Protein Block Serum-Free Ready-to-Use solution (DakoCytomation Japan, Kyoto, Japan) to reduce the non-specific background. The slides reacted with specific polyclonal antibodies against CD3 (1∶100, ab5690; Abcam, Cambridge, UK), CD31 (ab28365; Abcam), interleukin (IL)-6 (1∶400, ab6672; Abcam), and regulated on activation, normal T cell expressed and secreted (RANTES) (1∶50, NBP1-19769, Novus, Littleton, CO, USA) or monoclonal antibodies against F4/80 (1∶10,000, CL8940AP, Cedarlane, Hornby, ON, Canada) at 4°C overnight. Following this, they reacted with peroxidase-based EnVision+ (DakoCytomation Japan) for 30 min at room temperature, and were developed in 0.05% 3,3′-diaminobenzidine containing hydrogen peroxide. All sections were counterstained with hematoxylin and eosin. A catalyzed signal amplification system (CSA-Dako–Cytomation) was used to enhance the detection of F4/80 antigen, according the to manufacturer's instructions.

Immunostaining was performed using 8-μm cryosections as previously described.^{16 (link)} Antibodies in this study were anti-ETV2 (N-term) (AP11311a, ABGENT), anti-ETV2 (C-term) (AB65825, Abcam), anti-ETV2(Internal)(LS-C61735, Isbio), anti-hCD31 (AB32457, Abcam), anti-CD31 (AB9498, AB28365, Abcam), anti-ki67 (AB156956, AB15580, Abcam), anti-CD34 (AB157304, Abcam), anti-VE-cad (AB7047, Abcam), anti-flk1 (#2478, CST), anti-nestin (ab6320, Abcam), anti-CD133 (AB19898, Abcam), anti-GFAP (AB7260, Abcam), anti-NeuN (SAB4300883, Sigma), and anti-Sox2 (AB97959, Abcam). Sections were mounted using Prolong Gold with DAPI (Life Technologies). In situ hybridization was performed as previously described.^{16 (link)}