Anti mhc

Paraffin sections from 16 primary ERMS archival tumor specimens and three normal muscles from National University Hospital (NUH) and KK Women’s and Children Hospital in Singapore were analyzed by IHC using anti-EHMT2 antibody (1:50 dilution, Cell Signaling) as described (Bhat et al., 2019 (link)). Negative controls were performed using secondary antibody only. Images were captured with Olympus BX43 microscope (Ina-shi, Nagano, Japan). Approval was obtained from the ethics committee (IRB) at NUS. For IHC on mouse xenografts, sections were incubated with anti-EHMT2 (1:200, Abcam), anti-H3K9me2 (Abcam), anti-Ki67 (Leica Biosystems), anti-active-ß-catenin (1:300; Merck Millipore), anti-MHC (#M4276 1:200 Sigma-Aldrich), and anti-DKK1 (#ab61034 Abcam) antibodies followed by biotinylated goat anti-rabbit/anti-mouse IgG (H+L) secondary antibody (Vector Laboratories). Sections were washed and incubated with Vectastain Avidin–Biotin Complex (Vector Laboratories) for 20 min at 37°C.

After removing the medium, the cells in the culture plate were washed with PBS and then fixed with 4% paraformaldehyde (PFA, ChemCruz) for 15 min at RT. After washing with PBS, the cell membranes were permeabilized with 0.05% Triton X-100 (BioShop) in PBS for 3 min at RT. To limit the possibility of nonspecific binding of antibodies, cells were first incubated in a 0.25% glycine solution in PBS (30 min, RT), and then in 3% BSA in PBS (blocking buffer, 1 h, RT). Between each step, cells were washed with PBS. Primary antibodies (anti-MHC, Sigma-Aldrich, M4276; anti-myogenin, Santa Cruz Biotechnology, sc-576; and anti-MyoD, Santa Cruz Biotechnology, sc-304) were diluted 200 times in blocking buffer and incubated overnight at 4 °C. The next day, excess antibodies were washed away and secondary antibodies: goat anti-rabbit IgG Alexa Fluor 568 and goat anti-mouse IgG Alexa Fluor 568 (Thermo Fisher Scientific, A11011 and A11004, respectively, dilution 1:400) were administered for 2 h in the dark at RT. The nuclei were stained with Hoechst 33,342 (2 µg/ml, Sigma-Aldrich). The staining was analysed under a Nikon Eclipse fluorescence microscope.