Immunofluorescence staining for endothelial cells was performed using standard protocols. In Brief, the cells were fixed with 4% paraformaldehyde for 30 min, penetrated with 0.3% Triton X-100 for 1 h, and then blocked with 5% bovine albumin for 30 min. Subsequently, cells were incubated with anti-caspase-1 antibody (Proteintech, Chicago, USA, 1:50, Cat. No.: 22915-1-AP) at 4 °C overnight, followed by incubation at 37 °C with an Alexa Fluor 594 donkey anti-rabbit secondary antibody (Abcam, USA, 1:1000, Cat. No.: ab150076) for 1 h. Nuclei were stained by DAPI (Beyotime, China) for 20 min. Then the cells were photographed under a laser scanning confocal microscope (Nikon, A1 PLUS, Tokyo, Japan).
Anti caspase1 antibody
Manufactured by Proteintech
Sourced in United States
The Anti-Caspase1 antibody is a research-use only product designed to detect the presence of caspase-1, a key enzyme involved in the inflammatory response. This antibody can be used in various immunoassay techniques to identify and quantify caspase-1 levels in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
A1 plus, by Nikon (1 mentions)
Alexa fluor 594 donkey anti rabbit secondary antibody, by Abcam (1 mentions)
Anti zo 1 antibody, by Proteintech (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Abcam (1 mentions)
Anti il 18, by Abcam (1 mentions)
Anti β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Anti il 1β antibody, by Abcam (1 mentions)
Anti nlrp3 antibody, by Cell Signaling Technology (1 mentions)
Anti occludin antibody, by Proteintech (1 mentions)
Anti tubulin antibody, by Proteintech (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Goat anti rabbit igg h l hrp, by Jackson ImmunoResearch (1 mentions)
Rabbit anti gapdh polyclonal antibody, by Proteintech (1 mentions)
Anti nlrp3 antibody, by Abcam (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
4 protocols using anti caspase1 antibody
1
Immunofluorescence Staining for Endothelial Cells
2
Western Blot Analysis of Intestinal Proteins
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The protein expression in intestine tissue (n = 6) was detected by Western blot as previously mentioned [20 (link)]. Briefly, RIPA lysis buffer was used to extract the total protein and nucleoprotein were extracted with RIPA lysis at 14000g for 15 min. The protein was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after determining the protein concentration. Then the proteins were transferred onto polyvinylidene difluoride (PVDF) membranes. The resulting blots were blocked with 8% skim milk and incubated with anti-occludin antibody (1:1000; Proteintech, China), anti-ZO-1 antibody (1:1000; Proteintech, China), anti-TXNIP antibody (1:1000; Proteintech, China), anti-NLRP3 antibody (1:1000; Cell Signaling Technology, USA), anti-Caspase1 antibody (1:1000; Proteintech, China),anti-IL-1β antibody (1:1000; Abcam, USA), anti-IL-18 (1:1000; Abcam, USA),anti-N-terminal of GSDMD(1:1000; Abcam, USA), anti-β-actin antibody (1:10000; Santa Cruz Biotechnology, USA) overnight at 4 °C. Then, the bolts were incubated with horseradish peroxidase-conjugated secondary antibodies (1: 10000; Abcam, USA) for 1 h at 37 °C. The proteins were detected with the chemiluminescence (ECL) system. The expressions of proteins were normalized to β-actin as a reference.
3
MDBK Cell Culture and BVDV Isolation
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Madin–Darby Bovine Kidney (MDBK) cells were cultured in Dulbecco’s Modified Eagle Medium/Ham’s F-12 medium (1:1) (Gibco, Grand Island, NY, USA) supplemented with 7% fetal bovine serum (Thermo Scientific, Waltham, MA, USA) at 37 °C in a humidified atmosphere containing 5% CO2.
The NCP-BVDV-BJ175170 isolate strain, which was isolated from blood samples from cows with suspected BVDV infections, was maintained in our laboratory. The BVDV BJ175170 isolate strains were utilized for all experiments and are represented by “BVDV” in this article.
FTA (purity > 99%) was purchased from Selleck (Houston, TX, USA), solubilized in 100% DMSO, and kept frozen as 10 mM stocks. The compounds were stored in small aliquots to prevent multiple freeze-thaws and were stepwise diluted to reach the desired concentration in 0.5% DMSO for all treatments. Anti-NLRP3 antibody and anti-ASC antibody, anti-Caspase-1 antibody, anti-Tubulin antibody (ProteinTech Group, Rosemont, IL, USA), and anti-BVDV E2-protein mouse monoclonal antibody (mAb) (VMRD, Pullman, WA, USA) were used in the study.
The NCP-BVDV-BJ175170 isolate strain, which was isolated from blood samples from cows with suspected BVDV infections, was maintained in our laboratory. The BVDV BJ175170 isolate strains were utilized for all experiments and are represented by “BVDV” in this article.
FTA (purity > 99%) was purchased from Selleck (Houston, TX, USA), solubilized in 100% DMSO, and kept frozen as 10 mM stocks. The compounds were stored in small aliquots to prevent multiple freeze-thaws and were stepwise diluted to reach the desired concentration in 0.5% DMSO for all treatments. Anti-NLRP3 antibody and anti-ASC antibody, anti-Caspase-1 antibody, anti-Tubulin antibody (ProteinTech Group, Rosemont, IL, USA), and anti-BVDV E2-protein mouse monoclonal antibody (mAb) (VMRD, Pullman, WA, USA) were used in the study.
4
Analyzing Inflammatory Pathway Proteins
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Total protein was extracted from aortic tissues using RIPA lysis buffer (P0013B, Beyotime), and the concentrations of the isolated total protein were measured using a BCA protein assay kit (PL212989, Thermo). The collected protein samples were separated by 10% SDS-PAGE, and then transferred to PVDF membranes (IPVH00010, Millipore). After blocked with 5% nonfat milk (0.75 g milk powder + 15 mL PBS) at 37 °C for 1 h, the membranes were incubated with anti-Caspase1 antibody (1:1000, ProteinTech, USA), anti-GSDMD antibody (1:1000, ProteinTech, USA), anti-NLRP3 antibody (1:1000, Abcam, USA) and anti-GAPDH rabbit polyclonal antibody (1:1000, ProteinTech, USA) overnight at 4 °C. After that, the membranes were incubated with the secondary antibody (Goat anti-rabbit IgG (H + L) -HRP, 1:5000, Jackson ImmunoResearch, USA) for 2 h. Protein bands were visualized by enhanced chemiluminescence.
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