Horseradish peroxidase hrp labeled goat anti rabbit igg

The protein was extracted with RIPA lysate and protease inhibitor 100:1, and the BCA kit was used for protein quantification. After adding the loading buffer, the protein was boiled and stored at − 20 °C. The preserved protein samples were electrophoresed on a 6% SDS-PAGE gel at 80V 120 min; then 250 mA 150 min, electro-transported to the PVDF membrane; the skimmed milk powder was blocked for 1.5 h with rabbit anti-ABCA1 (1:1000, CST, USA), ABCG1 (1:500, Proteintech, Wuhan, China), SR-BI (1:500, Sangon Biotech, Shanghai, China) and GAPDH (1:1000, Sangon Biotech, Shanghai, China). Incubate overnight in a shaker at 4 °C, add horseradish peroxidase (HRP) labeled goat Anti-rabbit IgG (1:1000, Beyotime, Shanghai, China), using chemiluminescence western blot detection system to detect protein expression. Using GAPDH as an internal reference, the development results were analyzed for gray data results.

A total of 2 × 10⁶ T cells were incubated with 200 μL of RIPA buffer system (Beyotime, Shanghai, China) for 10 minutes. The cell lysate was centrifuged to collect proteins, which were separated through SDS-PAGE, after which Western blot analysis was performed. The primary antibodies for CD34, CD3ζ, and GAPDH included rabbit anti-CD34 (CST, USA), mouse anti-CD3ζ (Abeam, UK), and mouse anti-GAPDH (Beyotime). The corresponding secondary antibodies were horseradish peroxidase (HRP) labeled goat antirabbit IgG (Beyotime) and goat antimouse IgG (Beyotime).

Thirty milligrams of the ceca was cut into pieces and lysed in 1 ml of RIPA buffer containing protease inhibitors and phosphatase inhibitors (Beyotime Biotechnology). Samples were homogenized on ice, centrifuged for supernatant at 12,000 rpm for 30 min at 4°C, and heated to 100°C for 5 min. Protein extracts resuspended in sample loading buffer were separated by electrophoresis through 12–15% polyacrylamide gels. Following electrophoretic transfer of proteins onto polyvinylidene difluoride (PVDF) membranes (Millipore), non-specific binding was blocked by incubation with 5% non-fat dry milk (Sangon Biotech Shanghai Co., Ltd.), and then membranes were incubated with primary antibodies anti-phospho-ERK1/2 and anti-phospho-JNK1/2 (1:1,000 dilution, Cell Signaling Technology); anti-NLRP3, anti-NLRC4, anti-Caspase-1, anti-Caspase-11, anti-GSDMD, and anti-histone H3 (1:1,000 dilution, abcam); anti-GAPDH (1:1,000 dilution, Boster); and anti-Tubulin (1:1,000, Beyotime) overnight at 4°C. Membranes were then washed and incubated with the horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:3,000 dilution, Beyotime) for 1 h at room temperature. Proteins were visualized using enhanced chemiluminescence (ECL) reagent (Meilunbio). The grayscale values of the bands were determined by ImageJ launcher broken symmetry software program (National Institutes of Health, Bethesda, MD, USA).

Dulbecco’s modified Eagle’s F12 Ham medium (DMEM/F12) and fetal bovine serum (FBS) were obtained from Invitrogen (Carlsbad, CA, USA). Quercetin (#PHR1488) and diquat (#45422) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against PARP-1 (#9532), Caspase-3 (#9661S), and LC3A/B (#4108S) were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against Bcl-2 (sc-492), Bax (sc-493), and Nrf2 (sc-722) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against Claudin-1 (#519000), Claudin-3 (#341700), Claudin-4 (#364800), Occludin (#404700), zonula occludens (ZO-1, #617300), ZO-2 (#389100), and ZO-3 (#364100) were obtained from Invitrogen (Carlsbad, CA, USA). Horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (#A0208), HRP-labeled goat anti-mouse IgG (#A0216), JC-1 and EdU detection kits were purchased from Beyotime Biotechnology (Haimen, China). GSH detection kit was obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Unless indicated, all other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).

For IHC, Vero cells grown in 24-well plate were infected with attenuated PEDV DR13. At 18 h post infection, the cells were fixed and permeabilized and then air dried and incubated with rabbit anti-SF polyclonal antibody (harvested in Section 2.3) at a dilution of 1:50 in a humidified chamber at 37 °C for 60 min. After three washes with PBST (PBS with 0.1% Tween-20), the cells were incubated for 50 min at 37 °C with the Horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (Beyotime Biotech Inc., Shanghai, China) at a dilution of 1:200 in PBST. The cells were again washed 3 times with PBST, followed by incubation for 4–5 min at room temperature in diaminobenzidine solution (Solarbio, Beijing, China). Cell staining was examined using Nikon light microscope (Nikon corporation, Tokyo, Japan).