After transfection of PEI/MC and BP NCDs/MC nanocomplexes, the digested cell suspension was transferred to the confocal culture dish. After PBS washing, 4% polyformaldehyde fixed 1h, after PBS washing, with 0.2% of Triton X-100 rupture in 1h at room temperature, with PBS washing three times, add a goat serum albumin (GSA, Shanghai, China, Beyotime), down closed 2h at room temperature, out of confining liquid washing after adding suitable amount of COL1A1 (1:200; Cambridge, UK, Abcam), cTnI (1:500; Cambridge, UK, Abcam). The antibodies in each petri dish were then placed in a wet box and incubated overnight at 4°C. And then incubated with secondary antibodies (Goat Anti-Mouse IgG H&L (Alexa Fluor 488); Goat Anti-Mouse IgG H&L (Alexa Fluor 647), Cambridge, UK, Abcam) and 4ʹ,6-diamidino-2- phenylindole (DAPI) for 1h at room temperature. After washed with TBST (Shanghai, China, Beyotime), the cells were visualized under a Nikon fluorescence microscope.
For immunohistochemistry, heart tissues were stained with CD31 (1:200; Cambridge, UK, Abcam) for 24h at 4°C and then incubated with secondary antibodies (Goat Anti-Mouse IgG H&L (Alexa Fluor 647), Cambridge, UK, Abcam) and DAPI for 1h at room temperature. Sections were observed under a universal Nikon fluorescence microscope.
For immunohistochemistry, heart tissues were stained with CD31 (1:200; Cambridge, UK, Abcam) for 24h at 4°C and then incubated with secondary antibodies (Goat Anti-Mouse IgG H&L (Alexa Fluor 647), Cambridge, UK, Abcam) and DAPI for 1h at room temperature. Sections were observed under a universal Nikon fluorescence microscope.