Histological Analysis of Cardiac Tissue

At the end of reperfusion, 1 mm³ of left ventricle was immediately cut and placed in 4% paraformaldehyde for fixation to prepare frozen sections. The rat heart tissue was soaked in 4% paraformaldehyde solution for more than 24h, dehydrated with ethanol and embedded in paraffin, and then sliced into 4-6μm thick slices. These slices were performed by routine hematoxylin-eosin (H and E) and mason staining as described in our previous studies [24 (link), 25 (link)]. And the immunohistochemical (IHE) staining was performed on the next adjacent slice with a heat-induced antigen-retrieval step. All tissue sections were deparaffinized and hydrated, and endogenous peroxidase was blocked by hydrogen dioxide. The tissue sections were incubated with primary antibody against NLRP3 (1:200; Abcam, Cambridge, UK) overnight at 4° C. The negative control was the primary antibody replaced by phosphate-buffered saline (PBS). After washing with PBS, the slides were incubated using 3,3′-diaminobenzidine tetrahydrochloride (DAB, ZSGB-BIO, Beijing, China) to visualize the antigen-antibody compound. Finally, images were obtained by using a light microscope (LM, BX51, Olympus Co., Japan).