The following were stained on paraffin embedded tissue sections (thickness 5 μm) for: CCR2, (1:200; Abcam, Cambridge, MA), FSP-1 (1:500; Millipore, Temcula, CA), Smooth Muscle Myosin Heavy Chain (SMMHC, 1:50; Abcam) and Tlr9 (InvivoGen, San Diego, CA). For CCR2+, FSP-1, SMMHC cell counts, positive cells were counted and totaled in 5 high power fields (hpf, 1000X) radially around the IVC wall or thrombus.15 (link), 25 (link) Trichrome staining in the human sections was done as described.22 (link) Vein wall collagen content was determined using our previously described Sirius Red method.18 (link), 27 (link)To visualize NET in thrombus sections, we stained for cit-H3 (1:500, Abcam) and co-stained with extracellular DNA, labeled with 1uM SYTOX (Invitrogen, Grand Island, NY) at room temperature. Slides were cover slipped with ProLong Gold with DAPI mounting medium (Invitrogen). Pictures were taken using a Nikon Eclipse E400 microscope 1000x equipped with a Nikon Digital Sight DS-U3 camera using the DAPI (nuclei), FITC (SYTOX) and Texas Red (Cit-H3) channels.
Cit h3
Manufactured by Abcam
Sourced in United States, United Kingdom
Cit-H3 is an antibody that recognizes histone H3 proteins when acetylated at the lysine 9 residue (H3K9ac). This modification is associated with actively transcribed genes and is often used as a marker of gene activation.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Prolong gold, by Thermo Fisher Scientific (2 mentions)
Dapi mounting medium, by Thermo Fisher Scientific (2 mentions)
Digital sight ds u3, by Nikon (2 mentions)
Anti gapdh antibody, by Proteintech (2 mentions)
Goat anti rabbit igg, by Abcam (2 mentions)
Avidin biotinylated peroxidase complex abc, by Vector Laboratories (2 mentions)
Depex, by Merck Group (2 mentions)
Goat serum, by Merck Group (2 mentions)
Elastase, by Abcam (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Smooth muscle myosin heavy chain smmhc, by Abcam (1 mentions)
Fsp 1, by Abcam (1 mentions)
Sm mhc, by Abcam (1 mentions)
Imagequant las 4000 mini, by Fujifilm (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Pro prep protein extraction solution, by iNtRON Biotechnology (1 mentions)
35 protocols using cit h3
1
Immunohistochemical Profiling of Venous Thrombosis
2
Quantifying NET Formation in Brain Tissue
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Brain tissues were harvested after perfusion from WT and NLRP3 KO mice in each PBS and LPS-injected group and ground using a strainer. The total proteins were obtained using PRO-PREP protein extraction solution (iNtRON Biotechnology, Seongnam, Korea) and analyzed following the previously used method (6 (link)). In brief, primary Abs, CitH3 (ab5103; Abcam), NLRP3 (# AG-20B-0014; AdipoGen, San Diego, CA, USA), and β-actin (#8457; Cell Signaling Technology, Boston, MA, USA), were used to detect the NET formation in the brain tissue. After overnight incubation with primary Abs, the membrane was incubated with a secondary Ab for 1 h at 25°C. The membrane was developed using Clarity Max Western ECL substrate (Bio-Rad, Hercules, CA, USA) and the ImageQuant LAS 4000 mini (Fujifilm, Tokyo, Japan). Each time, the membrane was rinsed thrice with TBS-T. The signals were quantified using Image J (36 (link)) software.
3
Quantifying NETosis in Synovial Tissue
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Synovial tissues were digested in Hank's Balanced Salt Solution (Thermo Fisher Scientific) containing 0.25% trypsin (Gibco) at 37°C. Treated neutrophils or embedded tissues were fixed in 4% paraformaldehyde for 15 min and permeabilized in 0.01% Triton X‐100 for 10 min at room temperature. Samples were then stained with citrullinated histone H3 (CitH3) (1:50 dilution; Abcam) and LYG6 antibodies (1:100 dilution; eBioscience). The nucleus was counter‐stained with DAPI. Images were captured by confocal microscopy (Zeiss) from five randomly fields. The results were expressed as the number of NETs/50 Ly6G‐positive cells.
4
Western Blot Analysis of Lung and Platelet Proteins
5
Quantification of vWF, cit-H3, and IL-6 in Cell Cultures
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Following treatments, culture supernatants and cells were collected; cells were lysed in mammalian cell lysis buffer (CelLytic M, Sigma) and their protein content quantified using the bicinchoninic acid assay, as previously described [83 (link),84 (link),85 (link)]. Levels of vWF, cit-H3, and IL-6 in each culture supernatant (100 μL), as well as vWF levels in cell lysates (100 μL containing 50 μg protein), were quantified by ELISA using human vWF (Abcam, Waltham, MA, USA), cit-H3 (Cayman Chemical, Ann Arbor, MI, USA), and IL-6 (Invitrogen) ELISA kits in accordance with the manufacturer’s protocols. Standard curves from human vWF, cit-H3, and IL-6 reference standards (provided with each kit) were used, respectively, to quantify vWF, cit-H3, and IL-6 levels in each sample. Data were analyzed using Student’s t-test (two-tailed) or analysis of variance, followed by Tukey’s multiple-comparison tests, as previously described [46 (link)].For all figures, data are presented as mean ± standard deviation.
6
Neutrophil Cell Death Pathway Analysis
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[(3S,4R)-3-amino-4-hydroxy-1-piperidinyl][2-[1-(cyclopropylmethyl)-1H-indol-2-yl]-7-methoxy-1-methyl-1H-benzimidazol-5-yl]-methanone (GSK484) was obtained from Cayman Chemical (Michigan, USA). Antibodies against PAD4, Ly6g, CitH3, Neutrophil Elastase (NE), bcl-2, and caspase-3 were purchased from Abcam (Cambridge, UK). An antibody against GAPDH and rabbit IgG was acquired from Cell Signaling Technology (Beverly, USA). Interleukin 1β, interleukin 6, and tumor necrosis factor α (TNF-α) ELISA kits were provided by Thermo Fisher Scientific (Waltham, USA). A terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay kit was purchased from Roche Life Sciences (Indianapolis, USA). A blood urea nitrogen (BUN) was obtained from Thermo Fisher Scientific (Waltham, MA). A creatinine detection kit was obtained from Nanjing Jian Cheng Scientific (Nanjing, China).
7
Neutrophil Isolation and Citrullinated Histone H3 Detection
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Neutrophils were isolated from bone marrow using a Neutrophil Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instructions. Freshly isolated neutrophils were suspended in RPMI-1640 (Gibco, MA) and seeded at a density of 6×105 cells per mL in 48-well glass-bottomed plates. After treatment with 10 mg/mL Klebsiella pneumoniae lipopolysaccharide (LPS; Sigma Aldrich) for 2.5 h at 37 °C, the cells were fixed in 2% paraformaldehyde, blocked with 3% bovine serum albumin, incubated with rabbit anti-citrullinated histone H3 (Cit H3; 1:1000; Abcam) overnight at 4 °C, and then incubated with Alexa Fluor 488 donkey anti-rabbit secondary antibody for 40 min at RT. The nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI; Abcam).
8
Staining and Imaging of Neutrophil Extracellular Traps in Thrombi
9
Visualizing NET Formation in Mouse Brain
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Two-photon imaging was performed to observe NET formation in the mouse brain after 24 h of 2.5 mg/kg, intravenous (i.v.) LPS injection. An anesthetized mouse was placed in a customized chamber, and the brain surgery was conducted prior to imaging as previously described (41 (link)42 (link)). NET-defining markers, which are NE Ab Alexa Fluor 488 (Ssc-55549 AF488; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and CitH3 (Cat# ab5103; Abcam), were intravenously inoculated to visualize the NET formation. For the fluorescence imaging of NETs, the CitH3 Ab was conjugated to Alexa Fluor 594 using Ab labeling kits, as per the manufacturer’s instructions (Invitrogen). The blood vessel was visualized by injecting wheat germ agglutinin (WGA) (Cat# 29028; Biotium, Eching, Germany). Acquired Images were analyzed with Volocity, Image J, and Imaris based on previous reports (18 (link)).
10
Western Blot Analysis of Epithelial-Mesenchymal Transition Markers
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Whole cell lysates were extracted using RIPA lysis buffer (#P0013C, Beyotime Institute of Biotechnology, Inc.) with protease inhibitor (Roche Diagnostics). The quantities of protein were determined using a bicinchoninic acid kit (#P0012, Beyotime Institute of Biotechnology). Protein samples of 100 µg per lane were loaded onto 10% SDS-PAGE gels and transferred onto nitrocellulose membranes (PALL) for 90 min. For immunodetection, the membranes were incubated with the following primary antibodies at 4°C overnight: E-cadherin (#ab76055, Abcam) at a 1:1,000 dilution, vimentin (#HPA001762, Sigma-Aldrich; Merck KGaA) at a 1:1,000 dilution, and cit-h3 (#ab5103, Abcam), GAPDH (#60004-1-Ig, Proteintech) at a 1:1,000 dilution. The fluorescence-conjugated secondary IRDye800 mouse (#962-32210) and rabbit antibodies (#962-32211) were purchased from LI-COR. Following incubation with secondary antibody at 1:7,500 dilution with gentle shaking at room temperature for 60 min, the membranes were scanned by Odyssey Infrared Imaging System (LI-COR Biosciences). Protein expression was quantified using Image-Pro® Plus software (version 6.0; Media cybernetics, Inc.). All experiments were performed at least 5 times.
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