Gtx129270

Piglet lung tissues were isolated and fixed in 10% formaldehyde for 24 h at room temperature. Fixed tissues were dehydrated, embedded in paraffin, and cut in 8-μm sections. For histopathology, sections were rehydrated and stained with hematoxylin and eosin (C0105S, Beyotime) according to the manufacturer’s instructions.
For immunohistochemistry, tissue sections from pig lungs were placed on silane-coated slides, deparaffinized, and rehydrated using decreasing concentrations of ethanol. The sections were then treated with cell and tissue staining kit reagents (CTS005, CTS002, R&D), according to the manufacturer’s instructions, after being incubated with 10 mM citrate buffer for heat-induced antigen retrieval. The following primary antibodies were used at the respective concentrations: rabbit anti-PRRSV N (1:200, GTX129270, GeneTex), rabbit anti-cleaved CASP1 (Asp297) (1:200, 4199s, CST), mouse anti-ASC (1:100, sc-514414, Santa Cruz), and rabbit anti-NLRP3 (1:200, bs-10021R, Bioss).

Marc-145 cells or pig tissues were lysed in RIPA buffer containing 1% (v/v) phenylmethylsulfonyl fluoride (PMSF) (Beyotime, Jiangsu, China). The antibodies and their dilutions were shown as follows, anti-CEBPB (GTX100675, GeneTex, Alton Pkwy Irvine, CA, USA, 1:1000), anti-PRRS virus Nucleocapsid (GTX129270, GeneTex, Alton Pkwy Irvine, CA, USA, 1:1000), anti-β-tubulin (GB11017B, Servicebio, Wuhan, China, 1:1000), anti-β-actin (AC026, ABclonal, Wuhan, China, 1:20 000). The protein expression levels were normalized to corresponding β-actin or β-tubulin. ImageJ software was utilized for the quantitative analysis of Western blot results.

Cell cultured in a 12-well plate were washed with PBS once. Then 0.1 ml of pre-cooled methanol was added to each well and fixed at 4 ℃ for 30 min, dried in the ventilator, washed three times with PBS, and dried. PRRS virus nucleocapsid protein antibody (Gene Tex-GTX129270, USA) diluted at 1:500 was added, 0.1 ml per well, and incubated at 37 ℃ for 30 min. After washing it three times with PBS, and it was dried, FITC-labeled Goat anti-Rabbit IgG antibody (TransGen Biotech, Beijing, China) in 1:100 dilution was added, 0.1 ml per well, and incubated in a 37 ℃ temperature box for 30 min away from light. The wells were washed three times with PBS, dried, and DAPI (Absin, Shanghai, China) dye solution was added at room temperature for 10 min, and slid the hole plate and use a fluorescence microscope to look at the image until it becomes clear.