Rats from the different groups (n = 3 in each group) were anesthetized with 10% chloral hydrate (500 mg/kg, intraperitoneal), and the whole body of each rat was perfused through the left ventricle with 4% paraformaldehyde (pH 7.4). The cortex and hippocampi were removed from the brain of each rat and fixed in 4% paraformaldehyde overnight at 4°C. After dehydration in a concentration gradient of ethanol solutions and dimethylbenzene, the brain tissue samples were embedded in paraffin and cut into 5-μm-thick cross sections. The specimens were then deparaffinized and blocked with bovine serum albumin. The following primary antibodies were used in this study: NLRP3 (1:200, Bioss, China), caspase-1 (1:200, Abcam, USA), IL-18 (1:200, Abcam, USA), and IL-1β (1:200, Abcam, USA). Incubation was performed overnight at 4°C. After incubation with secondary antibodies at 37°C for 30 min, the sections were stained with diaminobenzidine and hematoxylin. Images were captured by microscopy (Zeiss, Germany). Ten images per rat were randomly selected, and at least thirty images per group were analyzed to determine the ratio of positive signal/image by using Image Pro Plus 6 software.
Nlrp3
Manufactured by Bioss Antibodies
Sourced in China
NLRP3 is a protein that plays a key role in the activation of the inflammasome, a multi-protein complex involved in the immune response. It serves as a sensor for various stimuli, including pathogen-associated molecules and endogenous danger signals, and its activation leads to the production of pro-inflammatory cytokines. NLRP3 is an important component in the regulation of innate immunity and inflammation.
Automatically generated - may contain errors
Lab products found in correlation
Caspase 1, by Abcam (2 mentions)
Nr1d1, by Bioss Antibodies (2 mentions)
Il 18, by Abcam (1 mentions)
Il 1β, by Abcam (1 mentions)
Ipvh20200, by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Gsdmd, by Bioss Antibodies (1 mentions)
Collagen 2, by Bioss Antibodies (1 mentions)
Mmp 13, by Bioss Antibodies (1 mentions)
Gadph, by ABclonal (1 mentions)
Il 1β, by ABclonal (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Rat elisa kit, by Neobioscience (1 mentions)
Anti caspase 1 antibody, by Santa Cruz Biotechnology (1 mentions)
Cell counting kit 8 cck 8, by 7Sea Biotech (1 mentions)
L nat, by Merck Group (1 mentions)
Anti asc, by Bioss Antibodies (1 mentions)
Nf κb, by Bioss Antibodies (1 mentions)
Il 1β, by Bioss Antibodies (1 mentions)
Brain derived neurotrophic factor (bdnf), by Bioss Antibodies (1 mentions)
8 protocols using nlrp3
1
Neuroinflammation and Apoptosis Evaluation
2
Quantitative Analysis of Innate Immune Receptors in Liver
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Liver tissues were stained with cross-reactive antibodies to selected PRRs, including RIG-I (Origene Technologies, Rockville, MD), TLR7 (Abcam, Cambridge, MA), TLR8 (Bioss, Woburn, MA), ZBP1 (Bioss), and NLRP3 (Bioss). Whole slide images were obtained using an Aperio Scanner GT450 (Leica Biosystems, Buffalo Grove, IL) and analyzed by QuPath, a quantitative pathology and bioimaging software developed at the University of Edinburg, UK (13 (link)). Five random areas of similar size were selected in each of the images and a set of same parameters was applied to each area for the detection of positively stained cells. The output results included the total number of immune and non-immune cells detected and the number of cells that stained positively. The percentage of stained cells in each whole slide image is presented as an average of the five areas. The number of positively stained immune cells in the same five areas was obtained by manual counting, a percentage was calculated based on the total number of cells detected and then averaged.
3
Protein Extraction and Analysis Protocol
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The Total Extraction Sample Kit was used to collect cell proteins. according to the manufacturer’s instruction, BCA protein assay kit (Beyotime, China, catalog no. P0011) was adopted to assess the protein concentration of different groups. Equal aliquots of the obtained protein were loaded and separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 5% Non-Fat milk was used to block the membranes for 1 h after electrotransfered onto PVDF membranes (Millipore, USA, catalog no. IPVH 20200), and then followed by incubation with primary antibodies overnight at 4°C. The primary antibodies used were as follows: NR1D1 (1:1000, Bioss, China, catalog no. bsm-33343M), NLRP3 (1:1000, Bioss, China, catalog no. bs-8878R), ASC (1:1000, Proteintech, USA, catalog no. 66444-1-Ig), caspase-1 (1:1000,abcam, China, catalog no. ab56416), IL-1β (1:1000, ABclonal, Wuhan, China, catalog no. A11025), GADPH (1:10000, ABclonal, Wuhan, China, catalog no. AC004), GSDMD (1:1000, Bioss, China, catalog no. bsm-33282M). After that, the membranes were incubated with the secondary antibodies for 1 h. The bands were captured by a computer imaging system. The expression level was semi-quantified by ImageJ software (NIH, USA).
4
Immunofluorescence Analysis of NLRP3 Inflammasome Pathway
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The P3 NPMSC were fixed with paraformaldehyde (4%) for 20 min. Then they were washed twice with PBS containing 0.5% Triton X-100 for 20 min. After that the cells were incubated with 15% bovine serum albumin for 2 h at room temperature, rinsed with PBS and incubated with primary antibodies: collagen II (Bioss, China, catalog no. bs-10579R) (1:200), matrix metalloproteinase 13 (MMP-13) (Bioss, China, catalog no. bs-10240R) (1:200), NR1D1 (1:1000, Bioss, China, catalog no. bsm-33343M), NLRP3 (1:1000, Bioss, China, catalog no. bs-8878R), ASC (1:1000, Proteintech, USA, catalog no. 66444-1-Ig), caspase-1 (1:1000,abcam, China, catalog no. ab56416), IL-1β (1:1000, ABclonal, Wuhan, China, catalog no. A11025), GADPH (1:10000, ABclonal, Wuhan, China, catalog no. AC004), GSDMD (1:1000, Bioss, China, catalog no. bsm-33282M) overnight (4°C). The second day, the plates were incubated with secondary antibody (1:500) (Bioss, China, catalog no. bs-0248M-FITC) for 2 h. The cell nucleus was labeled by DAPI for 5 min and captured under fluorescence microscope.
5
Investigating Inflammatory Pathways in Cellular Assays
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L-NAT was obtained from Sigma Aldrich (St. Louis, MO., USA). Rat ELISA kit was purchased from NeoBioscience (Shanghai, China). The cell counting kit-8 (CCK-8) came from 7sea-Biotech (Shanghai, China). Anti-ASC, NLRP3, IL-1β, TLR4 and NF-κB were purchased from Bioss Antibodies (Beijing, China). Anti-Caspase 1 antibody came from Santa Cruz Biotechnology (Shanghai, China). TRIzol reagent was obtained from Life Technologies (USA). RTPA lysis was purchased from SolarBio Life Sciences (Beijing, China).
6
Immunofluorescence Staining of NLRP3 and BDNF
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The cells were fixed in 4% paraformaldehyde, permeabilized, and blocked in 10% serum with 0.01% triton for 1 h at room temperature. The samples were then incubated with primary antibodies against NLRP3 (1:200, Bioss, China) and BDNF (1:200, Bioss, China) overnight at 4°C. An undiluted Alexa Fluor® 568 (red)-conjugated goat anti-rabbit IgG or Alexa Fluor® 488 (green)-conjugated anti-rabbit IgG secondary antibody was used. Images were captured with a Zeiss microscope and quantified with Image Pro Plus 6 software.
7
Acid-Sensing Ion Channel 1a and Chondrocyte Function
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The primary articular chondrocytes were pre-incubated as previously described. The total protein was extracted from a cell using RIPA protein lysates (150 mmol/l NaCl, 1.0% Nonidet P-40, 0.5% deoxycholate, 0.1% sodium dodecylsulfate (SDS), 50 mmol/l Tris and pH 8.0), and the supernatant of the mixture from each group was quantified by a fluorescent protein quantitative instrument (NanoDrop 2000, Thermo). The total protein samples from each group were separated by 10% SDS polyacrylamide gel electrophoresis and then blotted onto a polyvinylidene difluoride (PVDF) membrane. The membrane was incubated with 5% skim milk for 3 h and then probed with specific primary affinity-purified antibodies against ASIC1a (Alomone Labs,1:1000), caspase-1 p10 (Bioss,1:500), ASC (Bioss,1:500), NLRP3 (Bioss,1:500), Agg (Cell Signalling Technology,1:1000), Col 2a (Bioss,1:500) and β-actin (Bioss,1:500) overnight, followed by incubation with secondary antibodies for 1 h. The PVDF membrane was
The role of Ca 2+ in acid-sensing ion channel 1a-mediated chondrocyte. . . exposed and photographed using a chemiluminescence ECL kit.
The role of Ca 2+ in acid-sensing ion channel 1a-mediated chondrocyte. . . exposed and photographed using a chemiluminescence ECL kit.
8
Molecular Mechanisms of TRIM32-Mediated Inflammasome Activation
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BAY 11-7082, bicinchoninic acid (BCA) kit, and penicillin/streptomycin were purchased from Beyotime (Shanghai, China). TRIzol reagent and DAPI were obtained from Sigma-Aldrich (USA). The cDNA First-Strand Synthesis Kit and SYBR Green Master Mix were purchased from Roche (Switzerland). LipoFiter 3.0 was obtained from Hanbio Biotechnology (Shanghai, China), and FBS was obtained from Sijiqing Biological Engineering Materials Co., Ltd (Hangzhou, China). Antibodies against TRIM32 were purchased from Bioss (China), NLRP3, NF-κB p65, phospho-NF-κB p65, cleaved caspase-1, ASC, IL-18, and IL-1β were purchased from Wanleibio (Shenyang, Liaoning, China), GSDMD-N and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibodies were purchased from ABclonal (Wuhan, China).
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