Anti hla dr

To prepare the cells for immunofluorescence staining, hAMSCs were washed with 1x DPBS (Sigma, D5773) and fixed with 4% paraformaldehyde (EMPROVE®, Germany, 1,040,051,000) for 5 min at 4 °C. The cells were then washed again with 1x DPBS and permeabilized with 0.1% Triton-X100 in 1x DPBS for 5 min at 4 °C. The following primary antibodies were used for staining: anti-CD44 (1:50), anti-CD73 (1:200), anti-CD90 (1:100), anti-CD105 (1:100), anti-CD34 (1:250), anti-CD11b (1:250), anti-CD19 (1:250), anti-CD45 (1:250), and anti-HLA-DR (1:250) (Invitrogen™). Differentiating cells were stained with Sarcomeric alpha actinin monoclonal antibody (EA-53) (Invitrogen, MAI-22863, 1:500) and Anti-Cardiac Troponin T (abcam, ab8295, 1:1000) primary antibodies, Goat Anti-Mouse IgG H&L secondary antibody (abcam, ab150113, 1:1000), and DAPI (Biotium, 40043, 1:1000) to visualize nuclei. The antibodies were diluted in PBS containing 1% bovine serum albumin (Sigma, Germany, A2153) and cells were incubated with the primary antibodies overnight at 4 °C. The cells were then washed with PBS. To observe the results of immunofluorescence staining in cardiac genes, we used Leica DM2500 Optical Microscope (Leica Microsystems, Germany) equipped with a Canon EOS 7D camera (Canon, Japan). As part of the cardiac gene expression analysis, human cardiomyocytes (Promocell, C-12810) were used as positive control.