Antifading medium

Brains were isolated and fixed in 4% paraformaldehyde (PFA) for 24 h and then cryoprotected in 30% sucrose solution in PBS 1X for additional 2 days. After fixation and cryoprotection procedures, brains were cut using Leica Cryostat in 30 μm thickness. Sections were transferred into a blocking solution containing 0.1% Triton X-100, 10% goat serum in PBS 1X for 1 h at RT. Then, sections were incubated at 4°C overnight with the primary antibody, rabbit anti-NeuN (26975-1-AP; Proteintech, Wuhan, China; 1:500) diluted in PBS 1X, 0.1% Triton X-100, and 10% goat serum. After washing with PBS 1X for 1 h, the sections were incubated with secondary antibodies (Proteintech, Wuhan, China) diluted in PBS 1X for 2 h at RT. Afterward, sections were washed by PBS 1X thoroughly for 1 h and mounted with an antifading medium (Solarbio, Beijing, China). For quantitative analysis of NeuN staining, the average fluorescence intensity was determined using ImageJ software (version 1.52a; National Institutes of Health).

Cells were prepared in 48-well plates and treated with LPS and CATH-2 as described above. After 6 h incubation, cells were washed three times with PBS and fixed in 4% paraformaldehyde (Sango Biotech, Shanghai, China) for 20 min at room temperature (RT). After three wash steps, cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min. Subsequently, cells were blocked with 5% Bovine Serum Albumin (BSA) in PBS for 30 min. Then, cells were stained with primary antibody containing anti-NLRP3 (Bioss, Beijing, China) and anti-ASC (Santa cruz, CA, USA) for 1 h at RT. After the wash steps, cells were incubated with Goat anti-mouse IgG (H&L) Alexa fluor 488 and Goat anti-rabbit IgG (H&L) Alexa fluor 594 (Abcam, UK) for 1 h. DAPI (Beyotime Biotechnology, Shanghai, China) was added for 5 min to visualize cell nuclei. Finally, cells were washed and maintained in antifading medium (Solarbio, Beijing, China). Cells were observed using the fluorescence microscopy (Olympus, Tokyo, Japan).

PEC and neutrophils were cultured in a 48-well plate and infected with P. multocida for 3 h. After infection, cells were washed three times with PBS and fixed in 4% paraformaldehyde (Sango Biotech, Shanghai, China) for 20 min at room temperature (RT). After three wash steps, cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min. Subsequently, cells were blocked with 5% Bovine Serum Albumin (BSA) in PBS for 30 min. Then, the cells were stained with primary antibody containing rabbit anti-mouse NLRP6 (bs-10440R) (Bioss) and mouse anti-rabbit ASC (sc-514414) (Santa Cruz, CA, USA) for 2 h at RT, rabbit monoclonal anti-mouse histone H3 (citrulline R17) (ab219407) (Abcam, Cambridge, UK). After the washing steps, cells were incubated with Goat anti-mouse IgG (H&L) Alexa fluor 488 and Goat anti-rabbit IgG (H&L) Alexa fluor 594 (Abcam, UK) for 1 h. DAPI (Beyotime Biotechnology, Shanghai, China) was added for 5 min to visualize the cell nucleus. Finally, cells were washed and maintained in antifading medium (Solarbio, Beijing, China). The cells were observed on the fluorescence microscope (Olympus, Tokyo, Japan). Image J was used to calculate the positive staining cells.