Fluorescent labeled antibodies

For immunostaining, samples were fixed in 4% PFA overnight, embedded in optimal cutting temperature compound (Leica, #4583) and cut at 10 μm per section with a Leica CM1860 microtome (Leica). Sections were incubated with primary antibodies overnight at 4 °C. The following primary antibodies were used: α-actinin (1:400, Sigma-Aldrich, A5044), N-cadherin (1:500, Sigma-Aldrich, SAB5700640). After washing in 0.25% TritonX-100 in PBS, sections were incubated with fluorescent-labeled antibodies (Molecular Probes, Invitrogen) for 2 h.
For cell staining, cultured myoblasts and myotubes were washed briefly with PBS and fixed in 4% paraformaldehyde for 20 min and then permeabilized with PBS containing 0.125% Triton X-100 for 30 min. After being washed 3 times with PBS, the cells were blocked with 5% BSA at room temperature for 30 min. The primary antibodies were applied at 4 °C overnight. The following primary antibodies were used: desmin (1: 50; Abcam, ab32362), Myosin (1: 100; Abcam, ab264490), Talin (1:50, Abcam, ab71333), and Vinculin (1:500, Sigma-Aldrich, V4139). After washing in 0.125% TritonX-100 in PBS, cells were incubated with fluorescent-labeled antibodies (Molecular Probes, Invitrogen) for 2 h and then visualized under a fluorescence microscope (Leica DM6000).

The spleens from mice in each group were collected at posttransplantation day 8, after being grinded and filtered; and after lysing of the red blood, the splenocytes were washed and dispensed for using. FACS analysis was performed to determine splenocyte phenotypes and donor-reactive antibodies as previously described [11 (link)]. All the fluorescent-labeled antibodies against CD3, CD4, CD8, CD11c, CD25, CD68, CD86, CD206, Foxp3, IgM, IgG, and MHC class II were purchased from either eBioscience (eBioscience, San Diego, CA, http://www.eBioscience.com) or BioLegend (BioLegend, San Diego, CA, http://www.biolegend.com).