Rat hearts were excised and fixed with 4% paraformaldehyde for 24 h. For immunohistochemistry, the heart was embedded in paraffin, and 4-µm sections were prepared. The sections were incubated with the retrieval repair solution in a water bath for 30 min after deparaffinization, followed by blocking of endogenous peroxidase with 3% H2O2 for 15 min. After incubation with goat serum for 2 h, sections were treated with a rabbit anti-CD68 antibody (1:200, Proteintech, Chicago, IL, USA) at 4 °C overnight. Finally, the sections were stained with 3,3'-diaminobenzidine (DAB; Sigma-Aldrich).
For immunofluorescence analysis, the heart was dehydrated with gradient sucrose (10%, 20% and 30%). The samples were frozen and cut into 12-µm slices and dried for 24 h. Samples were incubated with primary antibody overnight at 4 °C, such as anti-Ccl2 antibody (1:200, EMD Millipore, Temecula, CA, USA), anti-CD206 antibody (1:200, Proteintech), anti-iNOS antibody (1:200, Proteintech), anti-CD14 antibody (1:200, Proteintech), and anti-CD11b antibody (1:200, ABclonal Technology Co.,Ltd, Wuhan, China). After being washed 3 times with PBS, the slices were stained with DAPI (4',6-diamidino-2-phenylindole, Sigma-Aldrich) and analyzed with a fluorescence microscope (Leica DMR 3000; Leica, Bensheim, Germany) to detect fluorescein isothiocyanate (FITC)-positive signals.
For immunofluorescence analysis, the heart was dehydrated with gradient sucrose (10%, 20% and 30%). The samples were frozen and cut into 12-µm slices and dried for 24 h. Samples were incubated with primary antibody overnight at 4 °C, such as anti-Ccl2 antibody (1:200, EMD Millipore, Temecula, CA, USA), anti-CD206 antibody (1:200, Proteintech), anti-iNOS antibody (1:200, Proteintech), anti-CD14 antibody (1:200, Proteintech), and anti-CD11b antibody (1:200, ABclonal Technology Co.,Ltd, Wuhan, China). After being washed 3 times with PBS, the slices were stained with DAPI (4',6-diamidino-2-phenylindole, Sigma-Aldrich) and analyzed with a fluorescence microscope (Leica DMR 3000; Leica, Bensheim, Germany) to detect fluorescein isothiocyanate (FITC)-positive signals.