Epr20829 408

Paraffin-embedded kidney sections were used to assess the co-localization of zonula occludens-1 (ZO-1) and GSDMD-N. First, the sections were subjected to microwave-based antigen retrieval using ethylene diamine tetraacetic acid antigen retrieval solution (pH 8.0), followed by tyramide signal amplification (TSA). Briefly, the following steps were performed: 1) incubation of sections with anti-GSDMD-N (1:200; Abcam; EPR20829-408) at 4°C overnight, 2) incubation with horseradish peroxidase (HRP) (1:500; Servicebio)-conjugated secondary antibody for 50 min at room temperature, 3) reaction with CY3-TSA (Servicebio, China) for 10 min in the dark, 4) removal of nonspecific binding antibodies by microwave treatment, 5) incubation with anti-ZO-1 (1:200; Servicebio) at 4°C overnight, 6) incubation with HRP (1:500; Servicebio)-conjugated secondary antibody for 50 min in the dark, 7) reaction with fluorescein isothiocyanate-TSA (Servicebio; China) for 10 min in the dark, and 8) staining with diamidine phenyl indole solution for 10 min. The images were captured by fluorescence microscopy using excitation wavelengths of 330–380 nm (blue), 510–560 nm (red) and 515–555 nm (green). The proportion (%) of positively stained glomerular area across three fields of view was analyzed using IPP. The results were confirmed by a professional pathologist.

In situ hybridization of formalin-fixed tissues and immunohistochemistry of paraformaldehyde-fixed tissues were performed as described [32 (link)]. The primary antibodies used for immunohistochemistry were against calprotectin (MAC 387; ThermoFisher Scientific), CD163 (GHI/61; Invitrogen), cytokeratin (AE1/AE3; Abcam), cleaved GSDMD (EPR20829-408; Abcam), histone H3 (ab18521; Abcam), IFN-1α (MMHA-2; Fisher), IL-1β (6E10; Novus Biologicals), MxA/Mx1 (4812; Novus Biologicals) and influenza A nucleoprotein (DPJY03, BEI Resources). To quantify expression in tissue, TIFF images taken by confocal microscopy were analyzed using Nikon NIS Elements version 4.50.00. Regions of interest were drawn to encapsulate lung tissue and exclude edges of tissue/airways in the tissue where no tissue is present. Microscope slides were imaged non-repetitively for 10 images per tissue section. Stains of interest were then quantified by recording the binary area (recorded as pixels) and averaging the values to generate individual data points per animal, which were averaged for presentation as single data points.

In all, 1 × 10⁶ BMDMs were either treated with 4HNE for 4 h or stimulated for 2 h with C. albicans yeast (MOI 10) followed by 4 h treatment with 4 HNE. The supernatant was discarded, and cells were incubated in 55 μl SDS-PAGE buffer. Samples were boiled at 95 °C for 5 min. The lysates were separated by SDS/PAGE, and the proteins were detected by immunoblotting with specific antibodies, including anti-pMLKL (S345) (D6E3G, Cell signaling; 1:1000), anti-MLKL (D6W1K, Cell signaling; 1:1000), anti-cleaved N-terminal GSDMD antibody (EPR20829-408, ab215203, Abcam; 1:1000), anti-rabbit IgG HRP (#7074, Cell Signaling; 1:10,000). To reprobe immunoblots, the membranes were stripped. The blots were developed using enhanced chemiluminescence and imaged with a C400 digital imager (Azure Biosystems). Uncropped raw immunoblots are presented in Fig. S20.