Comprehensive Immunohistochemical Analysis of Heart Tissue

Immunohistochemistry was performed as described previously^{31 (link)–33 (link)}. Briefly, heart tissue sections were permeabilized with PBS containing 0.5% Triton X-100 for 15 min at room temperature. Tissue sections were then blocked with 1% BSA in PBS for 60 min at room temperature and incubated with primary antibodies at 4 ℃ overnight. On the next day, samples were washed three times with PBS and incubated with secondary antibodies for 1h at room temperature. DAPI solution (VectaShield) was added for nuclear staining. The primary antibodies used in this study included mouse anti-Vimentin (Millipore; 1:100), rabbit anti-α smooth muscle actin (Abcam; 1:100), anti-cardiac Troponin T (Thermo; 1:100), rabbit anti-CD31 (Abcam; 1:100), mouse anti-CD68 (Abcam; 1:200), rabbit anti-CD206 (Abcam; 1:200), mouse anti-α sarcomeric actinin (Sigma; 1:100), and mouse anti-GFP (Thermo; 1:100). Secondary antibodies used in this study included either anti-mouse/-rabbit IgG Alexa Fluor 488 (Invitrogen; 1:500) or anti-mouse/-rabbit IgG Alexa Fluor 568 (Invitrogen; 1:500). Imaging of heart sections was performed using a Laser Scanning Microscope LSM 880 NLO with Airyscan (Zeiss).