Tunel dye

Mouse corneal sections and HCECs on 6-chamber slides were fixed with 4% paraformaldehyde (Solarbio, #P1110) and permeabilized with 0.2% Triton X-100 (Sigma–Aldrich, #9036-19-5) at room temperature for 10 min. The samples were then incubated with NLRP3 (1:50, Novus Biologicals, #NBP2-12446SS), ASC (1:50, Santa Cruz Biotechnology, #sc-514414), CASP1 (1:200, ABclonal, #A0964), GSDMD (1:100, ABclonal, #A18281) and Ly-6G antibodies (1:200, Servicebio, #GB11229) at 4°C overnight, followed by incubation at room temperature with secondary antibodies (1:300, Servicebio, #GB21303) for 1 h. For TdT-mediated dUTP nick end labeling (TUNEL) staining, the sections were incubated with TUNEL dye (Beyotime, #1086) for 30 min under shade. Nuclei were labeled with DAPI for 10 min. Finally, the samples were observed and photographed with a Leica TCS SP5 confocal microscope (Leica, Germany). For further detection the pyroptotic cells in corneal tissues, double-immunofluorescence staining of active CASP1 and TUNEL was performed on corneal sections. Active CASP1⁺/TUNEL⁺ cells were determined as pyroptotic cells (24 (link)).

Paraffin-embedded cardiac tissues were sectioned into 5 μm slices. After dewaxing to water, 20 μg/mL proteinase K was added to cover the tissue. Twenty minutes later, the sections were washed with PBS three times for 5 min, and TUNEL dye (C1086, Beyotime Biotechnology, Shanghai, China) was added and incubated at 37°C for 1 h. The slices were then washed with PBS three times for 5 min. DAPI was added to slices and incubated for 10 min. The slices were sealed with an antifluorescence quenching sealant. Fluorescence microscopy was performed in a darkroom for observation and image collection.

Heart tissues of rats were paraffin-embedded, sliced into 5 μm sections, and deparaffinized. The sections were treated with an autofluorescence quencher and 20 μg/mL of proteinase K to make the sample permeable, and washed with PBS for 5 min. The sections were incubated with TUNEL dye (C1086, Beyotime Biotechnology, China) at 37 °C for 1 h. After washing three times with PBS, the nuclei were counterstained with DAPI for 10 min, and the sections were immediately covered with autofluorescence quenching sealant. All sections were scanned using CaseView 2.0 (Pannorama 250/MIDI, 3DHISTECH, Budapest, Hungary).