3 3 diaminoben zidine

The sections at a thickness of 5 µm were dewaxed, immersed in sodium citrate buffer, incubated at 100°C for 2 min in an autoclave, and then rinsed 3 times with PBS to retrieve the antigens. Then, the sections were placed in 3% H₂O₂ and incubated at room temperature for 25 min in the dark to block the endogenous peroxide. The sections were sequentially incubated overnight with rabbit anti-human CDC25A (1:2,000; cat. no. GB11283; Wuhan Servicebio Technology Co., Ltd.) and rabbit anti-human IL-6 (1:600; cat. no. GB11117; Wuhan Servicebio Technology Co., Ltd.) antibodies at 4°C, and then with a goat anti-rabbit immunoglobulin G solution (1:200; cat. no. G23303; Wuhan Servicebio Technology Co., Ltd.) for 50 min at room temperature. The immunocomplexes were visualized using 3,3′-diaminoben-zidine and a light microscope (Olympus Corporation) at a magnification of ×200. Two blinded pathologists independently assessed all specimens. The staining intensity was scored as follows: 0 (negative); 1 (weak); 2 (moderate); and 3 (strong). The positive range scores were defined as follows: 0 (0-20%); 1 (21-50%); 2 (51-80%); and 3 (81-100%). The final score was obtained by multiplying the intensity score and the positive range score, and a score ³4 was regarded as high expression.

To direct observe virus particles in the mouse heart tissues, the heart paraffin sections were stained by immunohistochemistry (IHC) assay using a rabbit anti‐CVB3 polyclonal antibody (1:100; Millipore; Cat No: 2234140) as the primary antibody.  Nonimmune goat serum was used as negative control. After incubation with the indicated antibody, the tissue sections were visualized by 3,3‐diaminobenzidine (Zhongshan) and observed under a light microscope (Olympus BX63).
Immunofluorescence (IF) staining was performed to identify the infiltrated macrophages in mouse myocardium by incubating the tissues with mouse anti‐CD68 (1:200; Santa Cruz; Cat No: sc‐52998) monoclonal antibody, followed by hybridization with Dylight 649 goat anti‐mouse IgG (1:1000; Invitrogen). The nuclei of the cells were stained with 4,6‐diamidino‐2‐phenylindole (1:1000; Beyotime). Images were acquired and analyzed using a Leica TCS SP5 laser confocal microscope.