Serum citrullinated histone H3 or citrullinated histone H4 or carbamylated protein–dsDNA (double-stranded DNA) complexes were quantified by ELISA. A 96-well plate was coated with rabbit polyclonal anti-citrullinated histone 3 (Abcam) or citrullinated histone 4 (EMD Millipore) or carbamylated lysine (Cell Biolabs) antibodies at 1:400 in phosphate-buffered saline (PBS) overnight at 4°C. Wells were washed and blocked with 1% bovine serum albumin (BSA) at room temperature for 1 hour. Diluted plasma (1:10) was added to the wells in (1% BSA) blocking buffer and incubated overnight at 4°C. The wells were washed three times and incubated with mouse monoclonal anti-dsDNA antibody (EMD Millipore) at 1:100 in blocking buffer. After washing three times, goat anti-mouse–conjugated horseradish peroxidase (HRP) antibody (Bio-Rad) was added to the wells in blocking buffer at 1:10,000. Wells were washed five times followed by the addition of tetramethylbenzidine (TMB) substrate (Sigma-Aldrich) and stop solution (Sigma-Aldrich). The absorbance was measured at 450 nm, and values were calculated as an optical density (OD) index. Assay was performed in duplicate.
Anti citrullinated histone 3
Manufactured by Abcam
Sourced in United States
Anti-citrullinated histone 3 is a lab equipment product that detects the presence of citrullinated histone 3, a post-translational modification associated with various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Tetramethylbenzidine tmb substrate, by Merck Group (1 mentions)
Stop solution, by Merck Group (1 mentions)
Alexa fluor 488 donkey anti rabbit, by Abcam (1 mentions)
Alexa fluor 647 donkey anti goat, by Abcam (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Anti bax, by Abcam (1 mentions)
Anti caspase 3, by Abcam (1 mentions)
Sybrpremixex 2, by Takara Bio (1 mentions)
Anti caspase 9, by Proteintech (1 mentions)
Primescipt rt master mix, by Takara Bio (1 mentions)
β actin antibody, by Cell Signaling Technology (1 mentions)
Anti myeloperoxidase, by Abcam (1 mentions)
3 protocols using anti citrullinated histone 3
1
Quantification of Serum Citrullinated Histones and Carbamylated Protein-DNA Complexes
2
Evaluation of Lung Inflammation and NETs
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The left lung lobes were fixed in 4% PFA for 48 hours and then routinely processed to paraffin blocks and sectioned at approximately 4μm. H&E staining of lung tissue sections was performed to evaluate the inflammatory response. We stained mucus and mucus-containing goblet cells in the bronchial epithelium with an AB-PAS staining kit (Sigma-Aldrich). To detect NETs in lung tissue, paraffin-embedded mouse lung sections were permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS) for 15 minutes and blocked with 2% donkey serum. The sections were then incubated with the primary antibodies—anti-mouse myeloperoxidase (1:100; R&D Systems) and anti-citrullinated histone 3 (1:150; Abcam) overnight at 4°C. Then slides were incubated for 2 hours with secondary antibodies: Alexa Fluor 488 donkey anti-rabbit (1:1000; Abcam) and Alexa Fluor 647 donkey anti-goat (1:1000; Abcam). We used 4′, 6-diamidino-2-phenylindole (DAPI) to detect DNA. Finally, slides were visualized using a confocal microscope.
3
Antibodies and Reagents for Western Blot
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Antimyeloperoxidase, anti-Bax, anticitrullinated histone 3, and anti-caspase-3 were purchased from Abcam (Cambridge, MA, USA); anti-caspase-9 was purchased from Proteintech (Proteintech Group, Inc., USA); and β-actin antibody was purchased from Cell Signaling Technology (Danvers, Massachusetts, USA); TRIzol was purchased from Invitrogen (Waltham, Massachusetts, USA); PrimeScipt™ RTMasterMix and SYBRPremixEX™II were purchased from TaKaRa (Japan), and sulfotanshinone sodium injection. Sangon Biotech (Shanghai, China) synthesized the primers for synthesis.
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