Cells were cultured in a confocal dish until 50%-60% confluency. Cells were washed with PBS and fixed with 4% paraformaldehyde. After rinsing with 1% Triton solution, cells were permeabilized for 10 minutes. Then, cells were blocked with 4% BSA for one hour. The corresponding NLRP3 antibody (Proteintech, Wuhan, China) and ASC antibody (Affinity, Cincinnati, USA) were added and incubated overnight at 4 °C. After incubation, appropriate fluorescent secondary antibodies were applied and incubated. DAPI staining solution (Beyotime, Shanghai, China) was added for 10 minutes. Anti-fluorescence quencher was added and cells were examined under a Leica DMi8 inverted fluorescence microscope (Leica, Wetzlar, Germany).
Nlrp3 antibody
Manufactured by Proteintech
Sourced in China
The NLRP3 antibody is a tool used in scientific research to detect and study the NLRP3 protein. NLRP3 is a component of the NLRP3 inflammasome, which plays a role in the immune response. The antibody can be used in various laboratory techniques, such as Western blotting, immunohistochemistry, and immunoprecipitation, to identify and quantify the NLRP3 protein in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Dmi8 inverted fluorescence microscope, by Leica (1 mentions)
Dapi staining solution, by Beyotime (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Dmem with high glucose, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated goat anti rabbit igg, by Proteintech (1 mentions)
Txnip antibody, by Proteintech (1 mentions)
Las 4000 mini, by Fujifilm (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Enhanced chemiluminescence plus kit, by Merck Group (1 mentions)
Anti nf κb p65 antibody, by Abcam (1 mentions)
4 protocols using nlrp3 antibody
1
Immunofluorescence Staining of NLRP3 and ASC
2
Detailed protocol for cell culture and analysis
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BA (≥98%) was purchased from Sichuan Xieli Pharmaceutical Co., Ltd. (Chengdu, China, C031B180101). Luria–Bertani (LB) medium was prepared with yeast extract (OXOID, LP0021), tryptone (OXOID, LP0042B), and sodium chloride (Shanghai Test, 10019318). Fetal bovine serum (Gbico, 1456384) and trypsin (Gibco, 2520-056) were obtained from Gibco (Invitrogen S.r.l., Milan, Italy). DMEM with high glucose was obtained from Gibco (Hyclone, SH30243.01). RNA and qPCR reagents were obtained from Vazyme (Nanjing, China). Finally, ELISA kits were purchased from 4A Biotech (Beijing, China). The NLRP3 antibody was purchased from proteintech (Wuhan, China), and other antibodies were obtained from CST (Boston, MA, USA). All other chemicals were of reagent grade.
3
Immunohistochemical Analysis of Brain Samples
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For immunohistochemical observation, the sections of the brain sample were deparaffinized, rehydrated, and treated with 3% H2O2, followed by the addition of citrate buffer. The blocked sections were incubated with TXNIP antibody (catalog number: 18243-1-AP, Proteintech, Wuhan, China), or NLRP3 antibody (catalog number: 19771-1-AP, Proteintech), followed by incubation with the HRP-conjugated goat anti-rabbit IgG (catalog number: SA00001-2, Proteintech). The sections were subsequently treated with a 3,3-diaminobenzidine substrate system. The slides were observed using an Olympus light microscope (BX51, Olympus corporation).
4
Western Blot Analysis of Inflammation Factors
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Total proteins from samples were extracted in RIPA lysis buffer (Beyotime Biotechnology, China). Nuclear proteins were extracted using a nuclear protein extraction kit (Thermo Scienti c). Following incubation for 1h and centrifugation at 12,000 g (4℃, 20min), the supernatant was collected. Then, protein samples were separated using 8-12% SDS-polyacrylamide gels (SDS-PAGE) and transferred to a polyvinylidene di uoride (PVDF) membrane (Millipore, Boston, MA, USA). After blocking in 5% non-fat milk for 1h (25℃), membranes were incubated with the following speci c primary antibodies at 4℃ overnight: anti-NF-κB p65 antibody (Abcam, ab86299), NLRP3 antibody (Proteintech, 19771-1-AP), ASC/TMS1 polyclonal antibody (ABclonal, A16672), caspase-1/P10 antibody (Proteintech, 22915-1-AP). After washing with TBS-T buffer, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-rabbit or anti-mouse, 1:2000; Beyotime Biotechnology) at room temperature (25℃) for 1h. The immunolabeled proteins were detected using the Enhanced Chemiluminescence Plus kit (Millipore, USA) with LAS-4000 mini (Fuji, Japan). The level of β-actin was used as a loading control.
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