Cells were washed with ice-cold PBS and lysed with RIPA buffer (50 mM Tris–HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate) containing protease inhibitor cocktail. The samples were separated by SDS-PAGE and then transferred to polyvinylidene difluoride (PVDF) membrane (Millipore). The PVDF membrane was blocked with 5% nonfat milk and incubated with antibody against H3-Cit (1:1000 dilution, Abcam), p67phox (1:1000 dilution, Abcam), p47phox (1:1000 dilution, Gene Tex) at 4 °C overnight. Then the blots were incubated with an HRP-conjugated secondary antibody (1:5000 dilution, Santa Cruz Biotech) for 1 h at room temperature. Immunoreactive bands were visualized by Super Signal West Pico chemiluminescent substrate (Thermo Fisher) using Tanon-5200 chemiluminescent imaging system (Tanon, China).
H3cit
Manufactured by Abcam
Sourced in United States, China, United Kingdom
H3cit is a recombinant protein product. It is a histone H3 protein with a site-specific citrullination modification at the arginine 2 (R2) residue.
Automatically generated - may contain errors
Lab products found in correlation
Sytox green, by Thermo Fisher Scientific (3 mentions)
Elisa kit, by Jiangsu Meimian (3 mentions)
Lsm 780, by Zeiss (2 mentions)
P67phox, by Abcam (1 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Insulin, by Wuhan Servicebio Technology (1 mentions)
Neutrophil elastase, by Abcam (1 mentions)
Helix np blue, by BioLegend (1 mentions)
Cytochalasin d, by Cayman Chemical (1 mentions)
Celltracker orange, by Thermo Fisher Scientific (1 mentions)
Lactoferrin, by Abcam (1 mentions)
Cellmask deep red, by Thermo Fisher Scientific (1 mentions)
Baclight green bacterial stain, by Thermo Fisher Scientific (1 mentions)
Cd11b, by Cytek Biosciences (1 mentions)
Mouse igg alkaline phosphatase, by Abcam (1 mentions)
Murine fc blocking antibody 2.4g2, by Cytek Biosciences (1 mentions)
Histopaque 1119, by Merck Group (1 mentions)
Dsdna, by Abcam (1 mentions)
19 protocols using h3cit
1
Western Blot Analysis of Histone H3 Citrullination and NADPH Oxidase Subunits
2
Histopathological Assessment of Diabetes and Colitis
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The pancreas and colon were fixed in 4% paraformaldehyde and embedded in paraffin (Servicebo, Wuhan, China). To assess histopathological signs of diabetes, consecutive sections (4 μm) were then stained with hematoxylinand eosin (H&E, Sangon Biotech, Shanghai, China) and scored for insulitis, in which 0 stood for no infiltration, 1 stood for periinsulitis, 2 stood for infiltration covering approximately half of the islet, 3 stood for infiltration covering approximately 75% of the islet and 4 stood for full insulitis. For colon sections, after H&E staining, three independent parameters were determined: the depth of injury (scores 0, 1, 2 and 3 for none, mucosal, mucosal/submucosal and transmural, respectively), muscle layer thickness, and the ratio of villus height/crypt depth. Sections were also stained with PAD4 (Proteintech, Wuhan, China), Ly6G (Servicebio, Wuhan, China), H3cit (Abcam) and insulin (Servicebio, Wuhan, China) and subsequent fluorescein-labeled secondary antibodies to display NET formation or insulin production. Histological staining of mucins and calculation of mucus thickness were conducted according to previous descriptions (26 (link)).
3
Immunohistochemical and Immunofluorescent Analysis of NETs
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Immunohistochemical staining of paraffin-embedded sections was performed by the avidin-biotin-peroxidase complex method. Briefly, after rehydration and microwave antigen retrieval, primary antibodies were applied, incubated at 4°C overnight, and followed with secondary antibody incubation (GeneTech) at 37°C for 30 minutes. Staining was performed with 3, 30-diaminobenzidine tetra hydrochloride and counterstaining was performed with Mayer's hematoxylin. Immunofluorescence staining of NETs components in paraffin-embedded sections was performed similarly. Sections were proceeded with rehydration and antigen retrieval, followed by elimination of auto-fluorescence. Primary antibodies were then applied, incubated at 4°C overnight, and followed with fluorescence-conjugated secondary antibody incubation and Hoechst33342 stain of nuclear. Primary antibodies used: H3cit (1:100, Abcam), IL-8 (1:50, R&D). Photographs of 5 representative fields were captured and analyzed using software ImageJ with identical setting. NETs (marked as H3cit) and IL-8 were evaluated as percentage of area covered by positive staining.
4
Immunofluorescence Staining of NETs and CRC Cells
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For NETs detection, 2×105 neutrophils were seeded on poly-L-lysine-coated coverslips in 24-well plates to form NETs as described above, and then fixed with 4% PFA for 20 minutes. Subsequently, coverslips containing NETs were permeabilized with 0.1% Triton X-100 for 15 minutes at RT, washed with phosphate-buffered saline, and blocked with PBS containing 1% bovine serum albumin for 1 hour at RT. NETs were stained with primary antibody in blocking buffer at 4°C overnight. After wash, NETs were stained with matched fluorescence-conjugated secondary antibodies (Jackson; 1:600) in blocking buffer and finally stained with Hoechst33342 for nuclear(1:1000). Slides were then mounted with Fluoro-gel (Beyotime) and observed under fluorescence microscopy. Images were analyzed with ImageJ software. A similar procedure was performed for immunofluorescence staining of CRC cells. Primary antibodies used: H3cit (1:100, Abcam), MPO (1:100, Abcam), NF-κB (P65) (1:500, CST).
5
Comprehensive Analysis of Neutrophil Biology
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Antibodies specific for Ly6G, F4/80, CD35, and rabbit immunoglobulin G (IgG) were from BioLegend; CD11b was from Tonbo; CD63 was from BD Biosciences; and MPO, H3Cit, lactoferrin, PR3, neutrophil elastase, Fas, dsDNA, and mouse IgG alkaline phosphatase were from Abcam. Streptavidin Alexa Fluor 488 was from BioLegend. Live/dead stain and DHR123, CellTracker Orange, CellTrace Violet, CellMask Deep Red, and BacLight Green Bacterial Stain were from Invitrogen; Helix NP Blue was from BioLegend. Murine Fc-blocking antibody (2.4G2) was from Tonbo. PMA and DNase I were ordered from Sigma-Aldrich. Cytochalasin D was purchased from Cayman Chemical. Paraformaldehyde was from Electron Microscopy Sciences. The neutrophil elastase activity kit was purchased from Abcam. Histopaque 1119 and Histopaque 1077 were from Sigma-Aldrich; Lympholyte was from Cedarlane Laboratories. Dulbecco’s modified Eagle’s medium (DMEM) and phosphate-buffered saline (PBS) that were from Gibco and fetal bovine serum (FBS) from Atlanta Biologicals were used for all tissue culture.
6
Detecting Neutrophil Extracellular Traps
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NETs were detected by immunofluorescence staining as previously reported [28 (link)]. Neutrophils were fixed, permeabilized and blocked after 3-h in vitro culture or stimulation. Cells were incubated with antibody against histone H3 citrulline R2 + R8 + R17 (H3Cit; Abcam) at 1:400 for 1 h at 37℃, followed by secondary antibody coupled with Alexa Fluor Dyes (Invitrogen) at 1:1000 for 1 h at room temperature. DNA was stained using 4′,6-diamidino-2-phenylindole (DAPI; Cell Signaling Technology, London, UK) at 1:2000 for 8 min at room temperature. Images were obtained with a confocal fluorescence microscope (Zeiss LSM 510 META; Carl Zeiss, Thornwood, NJ, USA). NETs were identified as structures positive for both histone H3Cit and DAPI staining.
7
Immunohistochemical Analysis of Neutrophil Extracellular Traps
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Chromogenic immunohistochemical staining was performed using the Discovery Ultra Automated Immunostainer (Ventana Medical Systems, Tucson, AZ, USA) by the RI-MUHC Histopathology platform. Pre-treatment and post-treatment TMAs were stained for NETs and PMNs with H3Cit (Cat#: ab5103, 1:100, Abcam), NE (Clone: 950334, 1:100, Novus Biologicals, Oakville, ON, Canada) and CD8 (Clone: SP57, 1:100, Roche Diagnostics). TMAs were scanned using the Aperio Slide Scanner and slides were analyzed using QuPath software V6. TMAs were analyzed following the script created to analyze CD3 using fast cell counts67 (link).
8
Immunohistochemical and Immunofluorescence Staining of HCC Samples
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Immunohistochemical staining in paraffin-embedded sections was performed by the avidin-biotin-peroxidase complex method. Briefly, after rehydration and microwave antigen retrieval, primary antibody 8-OHdG (1:150, StressMarq Biosciences) was applied, incubated at 4°C overnight, and followed with secondary antibody incubation (GeneTech) at 37°C for 30 minutes. Staining was performed with 3,30-diaminobenzidine tetra hydrochloride and counterstaining was performed with Mayer's hematoxylin. Immunnofluorescence staining of NETs in resected HCC samples was performed in frozen section by 4% PFA fixation, incubation of primary antibody H3cit (1:150, Abcam) and 8-OHdG (1:150, StressMarq Biosciences), and fluorochrome-conjugated secondary antibody incubation with Hoechst 33342 stain of nuclear.
9
Quantifying Inflammatory Markers in Mice
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The concentrations of MPO-DNA complexes and NE in mice’s peripheral blood were assessed using an ELISA kit (MEIMIAN, China; Abcam, USA). Serum cf-DNA levels were determined after reacting with SYTOX green (ThermoFisher Scientific, USA) by comparing its absorbance to that of standard calf thymus (Sigma, USA). Additionally, a western blot of retinal proteins was conducted to analyze the citrullinated histone H3 (H3cit) and MPO (Abcam, USA).
10
Myocardial Infarction Size Evaluation
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To evaluate MI size, serial sections (10–12 sections per mouse, 400 µm apart, up to the mitral valve) were stained with Gomori’s 1-step trichrome stain. The infarcted area was determined for all sections using Diskus software (Hilgers, Königswinter, Germany) and expressed as a percentage of total left ventricular volume. Blue-stained collagen content was analyzed with Cell P Software (Olympus, Hamburg, Germany) and expressed as a percentage of the infarcted area. Serial sections (3 sections per mouse, 400 µm apart) were stained to analyse the infarcted area for neutrophil content (specific esterase, Sigma, St. Louis, MO), neutrophil extracellular trap formation (H3cit, abcam 5103, Cambridge, UK), macrophage content (F4/80), lymphocytes (CD3, both Serotec, Oxford, UK) apoptotic cells (TUNEL, MEBSTAIN apoptosis kit II, MBL, Woburn, MA, USA) and CD31-positive capillaries (Santa Cruz, Santa Cruz, CA). Positive-stained cells or vessels were counted in six different fields per section and expressed as cells or vessels per mm2.
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