Rat and human IVD tissues were obtained, fixed with 4% PFA for 48 h and then decalcified and embedded in paraffin. The tissues were cut into 5-μm-thick sections that were successively dewaxed and hydrated with xylene, anhydrous ethanol and an alcohol gradient (95%, 85% and 75%). The sections were then subjected to antigen retrieval with pepsin for 20 min at 37°C, incubated with neutralized endogenous peroxidase in a humidified box for 15‒20 min and sealed with goat serum for 30 min. The slides were then incubated overnight at 4°C with primary antibodies including anti-p-p65 (1:100; ABclonal), anti-HMGB1 (1:100; ABclonal), anti-NLRP3 (1:1000; Cell Signaling Technology), anti-caspase-1/p20 (1:100; Affinity), and anti-p21 (1:100; Santa Cruz Biotech, Santa Cruz, USA). The sections were subsequently rewarmed and incubated with biotin-labeled goat anti-mouse/rabbit secondary antibodies for 20‒30 min at room temperature and HRP-conjugated streptavidin (ZSGB-BIO, Beijing, China) for 25 min. DAB (ZSGB-BIO) staining and hematoxylin staining were performed for 1 min. Finally, images were captured using an Eclipse 80i fluorescence microscope (Nikon, Tyoko, Japan).
Anti nlrp3
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom, China
Anti-NLRP3 is a laboratory reagent produced by Cell Signaling Technology. It is an antibody that binds to the NLRP3 protein, which is a component of the NLRP3 inflammasome complex involved in the inflammatory response. The antibody can be used to detect and study the NLRP3 protein in various experimental applications.
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Lab products found in correlation
Anti il 1β, by Cell Signaling Technology (15 mentions)
Anti β actin, by Cell Signaling Technology (14 mentions)
Anti caspase 1, by Cell Signaling Technology (14 mentions)
Anti asc, by Santa Cruz Biotechnology (13 mentions)
Anti asc, by Cell Signaling Technology (12 mentions)
Anti gapdh, by Proteintech (10 mentions)
Nigericin, by Merck Group (8 mentions)
Anti cleaved caspase 1, by Cell Signaling Technology (8 mentions)
Anti mouse caspase 1, by Adipogen (7 mentions)
Mouse anti il 1β, by Cell Signaling Technology (7 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (7 mentions)
Adenosine triphosphate (atp), by Merck Group (7 mentions)
Mcc950, by Targetmol (7 mentions)
Ag 20b 0042, by Adipogen (7 mentions)
Sc 22514 r, by Santa Cruz Biotechnology (6 mentions)
Anti il 1β, by Abcam (5 mentions)
Anti cleaved il 1β, by Cell Signaling Technology (5 mentions)
Anti gsdmd, by Cell Signaling Technology (5 mentions)
Lps escherichia coli 055 b5, by Merck Group (5 mentions)
Epimedin a, by Targetmol (5 mentions)
89 protocols using anti nlrp3
1
Immunohistochemical Analysis of Inflammation in IVD
2
Microglia Protein Expression Analysis
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Microglia cells were seeded in 6-well plates. First, adherent cells were washed twice with 1 X PBS and collected by centrifugation at 600 g. The precipitate was lysed with 100 μL RIPA lysis buffer (containing 10 μL protease inhibitor cocktail tablets, each tablet dissolved in 1 mL RIPA solution, Roche, Mannheim, Germany). Lysates were centrifuged at 12,500 g for 15 min at 4°C to obtain supernatants of the whole cell lysates. Protein concentration was measured using a bicin-choninic protein assay (Beyotime Biotechnology, Shanghai, China). Proteins were electrophoresed through a 10–15% SDS-polyacrylamide gel and transferred to a polyvinylidene-difluoride (PVDF) membrane (Bio-Rad, Billerica, MA, USA). Blots were probed with the following primary antibodies (Cell Signaling Technology, MA, USA): anti-NLRP3 (1:1000), anti-caspase-1 (1:500), anti-β-actin (1:1000) and anti-IL-1β (1:500). The membranes were then incubated with enhanced chemiluminescence reagent (ECL) solution for 5 min. Immunolabeled bands were visualized using autoradiography.
3
Western Blot Analysis of NLRP3 Inflammasome Activation
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The lysates from cultured cells were centrifuged, the supernatant was collected, and total protein concentration was quantified by the Bradford protein assay (Bio-Rad, Hemel Hempstead, UK). The protein extracts (80 µg per sample) underwent SDS-polyacrylamide gel electrophoresis and were then transferred to a polyvinylidene difluoride membrane. The membranes were blocked with 5% non-fat dried milk and probed with primary antibodies: anti-NLRP3 (1:1000, Cell Signaling Technology), anti-ASC (1:500, Santa Cruz), anti-caspase-1 (1:1000, Abcam) and anti-GSDMD (1:1000, Novus Biologicals) in TBS-T overnight at 4 °C, followed by HRP-conjugated secondary antibody for 1 h. The loading control was GAPDH (1:10,000, Millipore). The blots were detected with enhanced chemiluminescence (ECL) system (Santa Cruz Biotechnology) and analyzed with GeneSnap (Syngene, Cambridge, UK). The protein band intensity was normalized with GAPDH and expressed as ratio of the control.
4
Antibody Acquisition for Autophagy and Inflammation
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Rabbit polyclonal anti-microtubule-associated proteins 1A/1B light chain 3A (LC3), anti-sequestosome 1 (SQSTM1)/p62, anti-NLRP3, anti-phospho-mechanistic target of rapamycin kinase (mTOR), anti-mTOR, and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology Inc. (Danvers, MA, United States). Rabbit monoclonal anti-LC3B, anti-SQSTM1/p62, anti-IL-1β, and Goat polyclonal anti-NLRP3 were purchased from Abcam (Cambridge, MA, United States).
5
Inflammatory Pathway Protein Analysis
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We purchased and used the following commercially available antibodies: anti-interleukin-1β, anti-ASC (apoptosis-associated speck-like protein containing a CARD), anti-caspase-1 (p10), anti-fibronectin (Santa Cruz Biotechnology, Inc., Dallas, TX), anti-NLRP3 (Cell Signalling Technology, Danvers, MA), anti-F4/80 (Bio-Rad Laboratories, Hercules, CA), anti-type I collagen (SouthernBiotech, Birmingham, AL), and anti-β-actin (protein loading control, Cell Signalling Technology).
6
Apoptosis and Inflammasome Signaling Assay
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The cell culture medium RPMI-1640, foetal bovine serum (FBS), and antibiotics (penicillin/streptomycin) were purchased from Gibco-BRL (Rockville, MD, USA). Fluorometric assay kit of caspase-3 activity was supplied by BioVision (Mountain View, CA, USA). 20,70-dichlorodihydrofluorescein diacetate, acetyl ester (H2DCF-DA) were purchased from Invitrogen. Anti-phospho-apoptosis signal regulating kinase (ASK), anti-ASK, anti-phospho-p38 mitogen-activated protein kinase (MAPK), anti-MAPK, anti-thioredoxin-interacting protein (TXNIP) and anti-β-actin antibodies were acquired from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-pro-caspase 1, anti-caspase 1, anti-NLRP3, anti-pro–interleukin (IL)-1β and anti-IL-1β antibodies were supplied by Cell Signaling Technology (Beverly, MA, USA). All other chemicals were supplied by Sigma-Aldrich (St. Louis, MO, USA).
7
Mtb-Induced Inflammasome Activation in THP-1 Monocytes
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THP-1 monocytes were seeded in 6-well plates at a density of 4.5 × 106 per well and incubated with 200 nm phorbol 12-myristate 13-acetate (PMA) for 72 h. Afterwards, the medium was changed to RPMI/FBS without PMA for 24 h. The cells were infected with Mtb Erdman at an MOI of 2 and supernatants were collected 24 h post infection. The supernatants were precipitated using methanol and chloroform and resuspended in Laemmli buffer. Proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis as previously described. The following antibodies were used: anti-ANT2 (14671; RRID: AB_2798562), anti-β-actin (4970), anti-cleaved IL-1β (83186; RRID: AB_2800010), and anti-NLRP3 (15101; RRID: AB_2722591) from Cell Signaling Technology and anti-GSDMD (HPA044487; RRID: AB_2678957) from Merck.
8
Inflammatory Signaling Pathway Analysis
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Carnosic acid, 5-aminosalicylic acid (5-ASA), sodium carboxymethyl cellulose, haematoxylin and eosin were purchased from Sigma Aldrich (St. Louis, MO). Dextran sodium sulfate was purchased from MP Biomedicals (Solon, OH). Anti-p65 (#8242), anti-phospho-p65 (#3039), anti-IĸBα (#9242), anti-phospho-IĸBα(#2859), anti-Stat3 (#4904), anti-phospho-Stat3 (#9145), anti-JNK (#9252), anti-phospho-JNK (#4668), anti-c-Jun (#2315), anti-phospho-c-Jun (#2361), anti-NLRP3 (#15101), anti-ASC (#67824), anti-Ubiquitin (#3933), iNOS (#13120), anti-H3k27Me3 (#9733), anti-H3k4Me3 (#9751) and anti-β-actin (#4970) antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-Keap1 (ab150654), anti-Nrf2 (ab62352) and Cullin3 (ab75851) antibodies were purchased from ABCAM. Anti-caspase1 (sc-56036) antibody was purchased from Santa Cruz Biotechnology, TRIzol reagent, TaqMan primers and TaqMan PCR Master Mix were purchased from Invitrogen (Carlsbad, CA).
9
Eh-rPrx Activates NLRP3 Inflammasome
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Following co-incubating native Prx or Eh-rPrx with RAW264.7 cells for 6 h, the cell lysate was collected for western blotting analysis. Primary antibodies used in this experiment included anti-β-actin (Abcam, UK), anti-NLRP3 (Cell Signaling Technology, USA), and anti-caspase-11 (Abcam, UK) antibodies; the secondary antibody used in this experiment was horseradish peroxidase-goat anti-rabbit IgG (H+L) (Abcam, UK). After co-incubating Eh-rPrx and RAW264.7 cells for 24 h, cell supernatant was collected. Similarly, cold acetone precipitation method was applied for protein extraction. The primary antibody used for western blotting was cleaved caspase-1 (Asp296) monoclonal antibody (Cell Signaling Technology, USA). ImageJ 1.52a (Wayne Rasband National Institutes of Health, Bethesda, MD, USA) was used for evaluating signal intensity.
10
Western Blot Analysis of NLRP3 and Caspase-1
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Lung samples and cells were homogenized in cell lysis buffer for western blotting. The protein concentrations were quantified with a BCA kit (Thermo Fisher Scientific). Equal amounts of protein (30 μg) were loaded, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis on 10% gels, and transferred to polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA). The membranes were blocked with 5% fat-free milk for 2 h at room temperature and then incubated with specific primary antibodies (anti-NLRP3, 1 : 2000 (Cell Signaling Technology, Danvers, MA, USA); anti-caspase-1 p20, 1 : 1000 (R&D, Minneapolis, MN, USA)) at 4°C overnight. After washing three times, the membranes were incubated with peroxidase-conjugated secondary antibodies (1 : 5000, Cell Signaling Technology) at room temperature for 1 h. Subsequently, bands were visualized with chemiluminescence (Millipore). The intensity of each band was analyzed using ImageJ (NIH, USA).
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