Primary antibody against nlrp3

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).

Brain tissue paraffin slides were heated at 65°C for 40 min, deparaffinized, and then rehydrated. The slides were immersed in citrate buffer (pH 6.0) and heated at 99°C for 18 min in a microwave oven for antigen retrieval, then cooled at room temperature. Endogenous peroxidase activity was quenched by treatment with 3% hydrogen peroxide (Zsbio, China) for 15 min at room temperature, and nonspecific binding sites were blocked by treatment with 10% normal goat serum (Zsbio, China) for 30 min at 37°C. Slides were incubated with primary antibody against NLRP3 (1 : 200, Abcam, USA) overnight at 4°C. The slides were subsequently incubated with biotin-labeled secondary antibody and streptavidin working solution (Zsbio, China) for 15 min. Next, the slides were visualized with 3,3′-diaminobenzidine (DAB) substrate chromogen system (Zsbio, China), rinsed with tap water, and counterstained with hematoxylin. Finally, immunoreactive signals were observed under a light microscope (Olympus, Tokyo, Japan).

Muscle samples were fixed in 4% paraformaldehyde at 4°C overnight. Then, they were conventionally prepared for paraffin embedding and sectioned into 4-μm slides. For the TUNEL assay, a One-Step TUNEL Assay Kit (Beyotime, Shanghai, China) was used. For immunofluorescence staining, tissue sections were boiled in 10 mM citrate buffer (pH 6.0) for 10 minutes and then processed with 0.25% Triton-100 for 30 minutes. A primary antibody against NLRP3 (1 : 250) was purchased from Abcam (Cambridge, UK). A caspase-1 antibody (1 : 50) was purchased from Santa Cruz (TX, USA). The sections were incubated in primary antibodies at 4°C overnight. The secondary antibodies were Alexa Fluor 488-conjugated donkey anti-mouse and Alexa Fluor 594-conjugated donkey anti-rabbit antibodies and were purchased from Jackson ImmunoResearch (PA, USA). Then, the sections were stained with DAPI for 5 minutes in the dark, and micrographs were taken with a Leica DM2500 (Wetzlar, Germany) microscope.