Protein expression was quantified using immunoblotting, as reported previously [27 (link),28 (link)]. Aortic tissues from the rats were rapidly isolated and frozen in liquid nitrogen. Aortic protein extracts (30 μg) were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Blots were incubated with anti-COX1 (~70 kDa; 1:1000, Cayman Chemical (#160109, Ann Arbor, MI, USA)), anti-COX2 (~72 kDa; 1:1000, Cayman Chemical (#160126, Ann Arbor, MI, USA)), and anti-β-actin (#A5316, 42 kDa; 1:5000) antibodies, and detection was achieved using horseradish peroxidase-conjugated immunoglobulin G followed by enhanced chemiluminescence. To normalize the data, we used β-actin as a housekeeping protein. The bands were analyzed using CS Analyzer 3.0 software (ATTO, Tokyo, Japan).
Anti cox 1
Manufactured by Cayman Chemical
Sourced in United States
Anti-COX-1 is a laboratory reagent used to inhibit the activity of cyclooxygenase-1 (COX-1), an enzyme involved in the synthesis of prostaglandins. This product is intended for research use only and its core function is to provide a tool for investigating the role of COX-1 in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti cox 2, by Cayman Chemical (6 mentions)
Anti β actin, by Merck Group (2 mentions)
Anti β actin, by Cayman Chemical (1 mentions)
Immun star western c kit reagent, by Bio-Rad (1 mentions)
Chemidoc xrs densitometer, by Bio-Rad (1 mentions)
Quantity one 1 d software, by Bio-Rad (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Lsm 510 meta microscope, by Zeiss (1 mentions)
Anti α sma, by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Dab substrate, by Agilent Technologies (1 mentions)
Envision system hrp labelled polymer, by Agilent Technologies (1 mentions)
Anti cox2, by BD (1 mentions)
Mayer s hematoxylin, by Agilent Technologies (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti pcna, by Santa Cruz Biotechnology (1 mentions)
Anti β catenin, by BD (1 mentions)
Anti phospho akt, by Cell Signaling Technology (1 mentions)
Anti claudin 3, by Thermo Fisher Scientific (1 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (1 mentions)
8 protocols using anti cox 1
1
Quantifying Aortic Protein Expression
2
Western Blot Analysis of COX-1 and COX-2 Proteins
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Cells were lysed with 250 μL of RIPA buffer [250 mM Tris/HCl, pH 8.0, 4.4 % NaCl, 5 % IGEPAL®, 2.5 % deoxycholic acid, 0.5 % sodium dodecylsulfate (SDS)]. 20 μg of proteins of total cell extract was loaded on 12 % polyacrylamide gel, and were separated by size using SDS polyacrylamide gel. Following electrophoresis, samples were transferred onto a nitrocellulose membrane and incubated with a primary monoclonal antibody anti-COX-1 (1:1000) and anti-COX-2 (1:1000) (Cayman®).
Blots were then incubated with a secondary peroxidase-conjugated antibody (1:10,000), and the primary antibody was performed. Protein bands were visualized by Immun-Star® Western C® Kit reagent (Bio-Rad Laboratories) Western blotting detection systems. The chemiluminescence signal was captured with a Chemidoc XRS densitometer (Bio-Rad Laboratories®) and analyzed with Quantity One-1D Software (Bio-Rad Laboratories®).
Blots were then incubated with a secondary peroxidase-conjugated antibody (1:10,000), and the primary antibody was performed. Protein bands were visualized by Immun-Star® Western C® Kit reagent (Bio-Rad Laboratories) Western blotting detection systems. The chemiluminescence signal was captured with a Chemidoc XRS densitometer (Bio-Rad Laboratories®) and analyzed with Quantity One-1D Software (Bio-Rad Laboratories®).
3
Platelet-Induced COX Expression in CASMCs
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CASMC (0.8 × 105 cells) were cultured alone or cocultured with platelets (0.5 × 108) for 8 h; then, cells were washed twice with phosphate buffer solution (PBS) pH 7.4 and fixed in acetone/methanol (40:60) for 20 min at room temperature. Cells were blocked with a filtrated solution of BSA (3%) in PBS for 30 min at room temperature. Cells were incubated overnight at 4 °C with polyclonal antibodies anti-COX-1 or anti-COX-2 (1:200, Cayman Chemical, Ann Arbor, MI, USA) and anti-α-SMA (1:200, Santa Cruz Biotechnology, Dallas, Texas, USA). Cells were then washed three times with PBS and incubated with the secondary antibodies, donkey anti-rabbit Alexa Fluor 488 and goat anti-mouse Alexa Fluor 546 (1:1000, Life Technologies, Waltham, MA, USA) for 1 h at room temperature. Cells were washed three times with PBS and incubated for 5 min with 4′,6-diamidino-2-phenylindole (DAPI; 300 nM, Sigma Aldrich) to label nuclear DNA. Finally, cells were washed and mounted in slides with Diamond antifade mounting media (Life Technologies). Slides were observed with a Zeiss LSM 510 meta microscope (Carl Zeiss, Jena, Germany), and confocal images were analyzed using Zeiss LSM 5 series software (Carl Zeiss).
4
Immunohistochemical Analysis of COX-1 and COX-2 in Mouse Growth Plates
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Growth plates were isolated from tibias of 6 weeks old C57BL/6 mice (approved by the local Animal Ethics Committee according to Dutch law; MUMC DEC approval 2008–042). Mice were euthanized using O2/CO2 asphyxiation. The tibias were fixed in formalin and decalcified in 0.5 M EDTA (Ethylenediaminetetraacetic acid) pH 7.8. Tibias were dehydrated, embedded in paraffin and five micrometer sections were cut. Sections were deparaffinized and antigen retrieval for COX-1 sections was performed by digestion with 0.4% hyaluronidase (Sigma-Aldrich, St. Louis, MO, USA) for 30 minutes at 37°C. For COX-2 detection, sections were incubated in hot citrate buffer for 30 minutes. Endogenous peroxidase activity was inactivated and sections were blocked with 10% normal sheep serum. Primary antibodies (anti-COX-1 (160110, Cayman Chemical, Ann Arbor, USA) and anti-COX-2 (610203, BD Transduction Laboratories)) were used in 1:100 dilution in PBS (Phosphate buffered saline). After washing in PBS-Tween20, bound antibodies were detected with HRP (horseradish peroxidase)-labelled secondary antibodies (Dako, EnVision+ System-HRP labelled Polymer). Bound secondary antibodies were visualized using DAB substrate (Dako, Glostrup, Denmark). Stained sections were counterstained with Mayer’s Hematoxylin (Dako), dehydrated and mounted in Histomount (Thermo Shandon).
5
Western Blot Analysis of Cell Signaling
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Cultured MOSE cells were washed with cold PBS and then lysed in SDS gel loading buffer. Lysates were collected and denatured in boiling water bath for 10 minutes. The protein extracts were separated on 8% or 10% SDS polyacrylamide gels, transferred onto nitrocellulose membrane, and subjected to immunoblotting according to standard procedures. The primary antibodies used were: anti–N-cadherin (mouse monoclonal, clone: 32/N-Cadherin, BD Bioscience), anti–E-cadherin (mouse monoclonal, clone: 34/E-Cadherin, BD Bioscience), anti–beta-catenin (mouse monoclonal, clone: 14/Beta-Catenin, BD Bioscience), anti-COX1 (#160105, #160110, Cayman Chemical), anti-COX2 (#160106, Cayman Chemical), anti–claudin-3 (Invitrogen), anti-PCNA (SC56, Santa Cruz), anti-p21 (mouse monoclonal, clone: SXM30, BD Bioscience), anti–phospho-AKT (#9276, Cell Signaling), anti-AKT (#9272, Cell Signaling), anti–phospho-Erk1/2 (mouse monoclonal, clone: E10, Cell Signaling), anti-Erk1/2 (#9102, Cell Signaling), and anti–beta-actin (clone AC-15, Sigma). Chemoluminescence detection was performed using the SuperSignal West Extended Duration Substrate detection kit (Pierce Biotech).
6
Lipopolysaccharide-induced Inflammation Assay
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Crystallized Calloselasma rhodostoma venom, lipopolysaccharides from Escherichia coli O111:B4 (LPS), Histopaque 1077, 3,30-diaminobenzidine tablets, hydrogen peroxide 30%, ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), HEPES, Triton X-100, leupeptin, aprotinin, phenylmethylsulfonyl fluoride (PMSF), sodium orthovanadate, protease and phosphatase inhibitor cocktail, ethylenediaminetetraacetic acid disodium salt dihydrate (Na2 EDTA), bicinchoninic acid protein assay Kit (BCA), oil Red O, AACOCF3 (arachidonyl trifluoromethyl ketone), anti-mouse Fab specific-FITC and anti-β-actin were purchased from Sigma Chem. Co. (Misssouri, USA). CAY10650, A922500, prostaglandin E2 ELISA Kit, anti-COX-1 and anti-COX-2 were purchased from Cayman Chemical (Michigan, USA). Anti-cPLA2-α, anti-PTGES and anti-p-cPLA2-α were purchased from Santa Cruz Biotechnology (Texas, USA). PureLink kit, SuperScript III Reverse Transcriptase, GeneChip WT PLUS Reagent Kit and GeneChip Clariom S Array Human were purchased from Thermo Fisher Scientific (Massachusetts, USA). iTaq Universal SYBR Green Supermix were purchased from Bio-Rad (California, USA). All salts and reagents used obtained from Merck Millipore (Darmstadt, Germany) with low endotoxin or endotoxin-free grades.
7
Antibody Sourcing for Neurological Assays
8
Protein Extraction and Western Blot Analysis
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Proteins from mouse abdominal aorta and mAECs were extracted with cold lysis buffer (50 mM Tris•Cl, pH 7.2, 1% (w/v) Triton X-100, 0.1% (w/v) SDS, 500 mM NaCl and 10 mM MgCl2) supplemented with protease and phosphatase inhibitors (Roche). Polyacrylamide gel-electrophoresis and western blot were performed as described [22] . The following primary antibodies were used at the indicated dilutions: anti--actin 1/2000 (sc-47778), anti ERK-2 1/1000 (sc-1647), anti-iNOS 1/1000 (sc-650), and anti-eNOS 1/1500 (sc-654) from Santa Cruz Biotechnology; anti-COX-1 1/1000 (160109) and anti-COX-2 1/1000 (160112) from Cayman Chemical. Secondary HRP-conjugated antibodies were from Santa Cruz Biotechnologies: anti-IgG-rabbit 1/5000 (sc-2004) and anti-IgG-mouse 1/5000 (sc-2005).
Immunocomplexes were detected by incubation with Luminata Forte Western HRP substrate (Millipore).
Immunocomplexes were detected by incubation with Luminata Forte Western HRP substrate (Millipore).
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