The paraffin sections of LGs (4 µm) were stained with H&E and subsequently observed with a microscope. The LG cryosections or primary LG epithelial cells were fixed with 4% paraformaldehyde (Solarbio, Wuhan, China). The LG cryosections were incubated with NLRP3 (1:200; Affinity Biosciences, Cincinnati, OH, USA), caspase-1 (1:200; ABclonal, Wuhan, China), and GSDMD (1:200; Affinity Biosciences), and the primary LG epithelial cells were incubated with AQP5 (1:200; Abcam, Cambridge, MA, USA), epithelial cell adhesion molecule (1:200; Affinity Biosciences,), NLRP3 (1:200; Affinity Biosciences), caspase-1 (1:200; ABclonal), and GSDMD (1:200; Affinity Biosciences), followed by fluorescein-conjugated secondary antibodies (1:200; Life Technologies, Grand Island, NY). The cryosections were visualized using a fluorescence microscope (Olympus, Tokyo, Japan).
Nlrp3
Manufactured by Affinity Biosciences
Sourced in United States, China
NLRP3 is a protein that serves as a key component of the inflammasome, a multi-protein complex involved in the inflammatory response. It functions as a sensor that detects various danger signals and triggers the activation of caspase-1, leading to the release of pro-inflammatory cytokines.
Automatically generated - may contain errors
Lab products found in correlation
Gsdmd, by Affinity Biosciences (6 mentions)
Caspase 1, by Affinity Biosciences (4 mentions)
P iκbα, by Affinity Biosciences (3 mentions)
Il 1β, by Affinity Biosciences (3 mentions)
β actin, by Affinity Biosciences (3 mentions)
Tnf α, by Shanghai Enzyme-linked (3 mentions)
Il 1β, by Shanghai Enzyme-linked (3 mentions)
Interleukin 6 (il 6), by Shanghai Enzyme-linked (3 mentions)
Il 10, by Shanghai Enzyme-linked (3 mentions)
Il 18, by Shanghai Enzyme-linked (3 mentions)
Tgf β, by Shanghai Enzyme-linked (3 mentions)
Ifn γ, by Shanghai Enzyme-linked (3 mentions)
4 hne, by Alpha Diagnostic (2 mentions)
Caspase 1, by Proteintech (2 mentions)
Il 18, by Affinity Biosciences (2 mentions)
Mmp13, by Proteintech (2 mentions)
Collagen 2, by Abcam (2 mentions)
Histone h3, by Affinity Biosciences (1 mentions)
Gapdh, by Affinity Biosciences (1 mentions)
Erythromycin, by Meilun (1 mentions)
17 protocols using nlrp3
1
Immunofluorescence Analysis of LG Inflammasome Pathways
2
Biochemical Assays and Reagents Protocol
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COP (purity >98%) was purchased from Shanghai Yuan Ye Biotechnology Co., Ltd (Shanghai, China). Dextra sulfate sodium was obtained from MP Biomedical (Irvine, CA, USA). Cefadroxil (purity >99%), terramycin (purity >98%) and erythromycin (purity >99%) were purchased from Dalian Meilun Biotechnology Co., Ltd (Dalian, China). Mesalazine (MSZ) was obtained from Losan Pharma GmbH, Germany. ELISA kits (TNF-α, IL-6, IL-10, IL-18, IL-1β, TGF-β, and IFN-γ) were obtained from Shanghai Enzyme-linked Biotechnology Co., Ltd (Shanghai, China). BCA kit and myeloperoxidase (MPO) assay kit were obtained from the Jiancheng Bioengineering Institute of Nanjing (Nanjing, Jiangsu, China). Primary antibodies against p65, IκBα, p-IκBα, NLRP3, ASC, Capase-1, Caspase-1 p10, GAPDH and Histone H3 were purchased from Affinity Biosciences (OH, USA). All other reagents and chemicals were at least of analytical grade.
3
Quantifying Oxidative Stress Biomarkers
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Skin tissue was lysed in RIPA lysis buffer and protein was collected under centrifugation at 12,000× g rpm for 15 min at 4 °C. Protein concentrations in skin tissues were measured. The protein sample was diluted with loading buffer and boiled at 100 °C for 5 min. The protein samples were then separated by SDS-PAGE (concentration: 10%). They were then transferred to the PVDF membrane. The membrane was sealed with blocking buffer (5% milk) for 1 h, and incubated overnight with primary antibodies in a 4 °C refrigerator. The primary antibodies included NLRP3, caspase-1, IL-1β, γH2AX, Nrf2 (1:1000, Affinity Biosciences, Cincinnati, OH, USA), 3-NT (1:1000, Millipore, Boston, MA, USA), 4-HNE (1:1000, Alpha Diagnostic International, San Antonio, TX, USA), HO-1, CAT, and anti-β-actin (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The membrane was then rinsed with TBST (0.05% Tween 20) three times and incubated with horseradish peroxidase-labeled secondary antibody for 1 h at room temperature. Finally, ECL kit was used to indirectly detect protein expression level. All experiments were repeated at least three times. Image J v1.8.0 software was used to conduct grayscale analysis of protein expression bands.
4
Molecular Analysis of Penile Inflammation
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The center portions of the rat penile shafts were severed and fixed in 4% paraformaldehyde. The samples were embedded and cut into 4-μm sections before mounting on glass slides. The penile tissue sections were incubated with primary antibodies to IL-18 (Affinity Bioscience; 1:800), NLRP3 (Affinity Bioscience; 1:1000), caspase-1 (Proteintech; 1:1000), GSDMD (Proteintech; 1:1000), and eNOS (Proteintech; 1:1000) overnight at 4 °C, while the control sections were incubated without the primary antibodies. Next, the sections were incubated with the goat anti-rabbit secondary antibodies at a 1:200 dilution for 2 hours at room temperature. Quantitative image analysis used computational densitometry using the ImageJ application (National Institutes of Health) and a microscope. The levels of marker expression in each were then normalized as a ratio to control. Ten sections of rat penile tissue were counted, and 6 fields were computed from each section.
5
Immunofluorescence Staining of Chondrocytes
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Immunofluorescence staining was conducted following standard protocols and our previous methods [19 (link)–21 (link)]. Chondrocytes that had undergone suitable treatment were fixed with 4% PFA for 30 minutes and permeabilized in 0.3% Triton X-100 at room temperature (RT) for 30 minutes. After incubating with 10% FBS for 1 hour at RT to block nonspecific binding, the samples were incubated overnight with primary antibodies against MYD88 (1:200, Abcam), collagen II (1:200, Abcam), MMP13 (1:200, Proteintech), NLRP3 (1:200, Affinity), GSDMD (1:100, Affinity), p-IκBα (1:200, Affinity), and p-p65 (1:200, CST) at 4° C. Next, the samples were incubated with secondary antibodies (1:300, Invitrogen). Finally, the fluorescence intensity of positive cells was observed under fluorescence microscope.
6
Inflammatory Signaling Pathway Analysis
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Group settings were consistent with the description in section 2.7 (“Inflammation modeling”). The detailed procedure for western blot assays is consistent with the description in Section 2.5. Source antibodies: IL-1β, TNF-α, TGF-β1, TLR4 (1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA); IL-6, IL-10, p705-STAT3, NLRP3 (1:1000; Affinity Biosciences, Jiangsu province, China); p38, p-p38, ERK, p-ERK (Cell Signaling Technology, USA); myD88, JNK, p-JNK, p65, p-p65 (Proteintech Group, Wuhan, China); β-actin (ABclonal, China).
7
Cardiomyocyte Protein Expression Analysis
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Heart tissues and AC16 cardiomyocytes were homogenized in ice-cold 1× RIPA lysis buffer, supplemented with a protein inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) to obtain total protein. Total protein was separated using 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 5% non-fat milk for 1 h and incubated overnight at 4 °C with the following antibodies: 3-NT (Millipore, Billerica, MA, USA), 4-HNE (Alpha Diagnostic International, SAN Antonio, TX, USA), NLRP3, caspase1, IL-1β, TXNIP (Affinity, Jiangsu, China) and β-actin (Santa Cruz, Dallas, TX, USA). After washing unbound antibodies, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz, Dallas, TX, USA) for 1 h at room temperature. Specific bands were visualized using an enhanced chemiluminescence detection kit (ECL) and Gel Documentation 2000 system (Bio-Rad, Hercules, CA, USA). Densitometric analysis of protein bands was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
8
Multiplex Immunoassay for Immune Profiling
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Commercial Nig, DAC were purchased from Yuanye Bio-Technology Co., Ltd. (Shanghai, China). His6 was acquired from GL Biochem Co., Ltd. (Shanghai, China). All the ELISA kits were acquired from Beyotime (Shanghai, China). Rabbit anti-mouse antibodies NLRP3, GSDMD, β-actin, and Ki67 were acquired from Affinity Biosciences (Beijing, China). Antibodies CD3, CD4, CD8 CD86, CD11c, CD45, F4/80, CD86, CD206 (MMR) were purchased from BioLegend (Shanghai, China).
9
Quantifying Protein Levels in BMDMs
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Following extracting total proteins from BMDMs with the lysis buffer, the bicinchoninic acid (BCA) kit (ZIKER, Guangdong, China) was utilized to quantify the concentration of proteins, which were then loaded and separated by the 12% SDS-PAGE [22 (link)]. The proteins were then transferred onto the PVDF membrane (Invitrogen, California, USA), incubated with 5% BSA, and then with the primary antibody against NOX-4 (1:1000, Affinity, Melbourne, Australian), IRT1 (1:1000, Affinity, Melbourne, Australian), NLRP3 (1:1000, Affinity, Melbourne, Australian), β-actin (1:1000, Affinity, Melbourne, Australian). After overnight incubation, the membrane was washed 3 times and incubated with the secondary antibody (1:2000, Affinity, Melbourne, Australian), followed by being exposed to the ECL solution. Lastly, the Image J software was utilized to visualize the bands.
10
MDP-Mediated Inhibition of NLRP3 Inflammasome
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MDP [C29H32O13S, molecular weight: 620], purity > 98%, was provided by the Chemistry Laboratory of the Institute of Clinical Pharmacology of Anhui Medical University (Hefei, China); LW6 (CAS: 934593-90-5) was obtained from MedChemExpress (USA). N-acetyl-L-cysteine (NAC) was purchased from Beyotime Biotechnology (Shanghai, China). Dulbecco's modified Eagle's medium (DMEM) was obtained from Gibco Co. Ltd. (CA, USA). Fetal calf serum was purchased from Wisent Co. Ltd. (Canada). Streptomycin, penicillin, ROS Assay Kit, and goat anti-rabbit IgG were purchased from Beyotime Biotechnology Co. Ltd. (Shanghai, China). Enzyme-linked immunosorbent assay (ELISA) kits for IL-1β and IL-18 were the products from J&L Biological (Shanghai, China). Antibodies against GRK2, p-GRK2 S670, HIF-1α, NLRP3, ASC, Caspase-1, GSDMD, and β-actin were purchased from Affinity Bioscience (Taiwan, China). The antibody against VHL was the product from Wanleibio (Shenyang, China).
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