Formation of NETs was quantified as in previous study [16 (link)]. Briefly, in in vitro experiments, NETs were identified with Sytox Green staining. Sytox Green (Invitrogen, 1:30000 diluted in Hanks’ Balanced Salt Solution, HBSS) was applied to neutrophils cultured on coverslips for 15min and washed with HBSS. In in vivo analysis of NETs formation, brain slices were collected and stained with rabbit anti-CitH3 (1:500, Abcam). Images were collected with a microscopy (Nikon). The recorded images were loaded onto Image J (NIH) for further analysis, at which time NETs were manually quantified by 2 blinded observers. Decondensed nuclei (stained with DAPI), which also positively stained with Sytox Green or CitH3 and displayed cloud-like morphology, were considered NETs and digitally recorded to prevent multiple counts. The percentages of NETs were calculated as the average of at least 4 fields, normalized to the total numbers of cells.
Rabbit anti cith3
Manufactured by Abcam
Sourced in United Kingdom
Rabbit anti-CitH3 is a primary antibody that recognizes citrullinated histone H3. It is designed for use in various immunoassay applications to detect and quantify citrullinated histone H3 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Sytox green, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 goat anti rat igg, by Thermo Fisher Scientific (1 mentions)
Imagelab 4.1 image analysis software, by Bio-Rad (1 mentions)
Mn protein quantification assay, by Macherey-Nagel (1 mentions)
Nylon nitrocellulose membranes, by Bio-Rad (1 mentions)
Rabbit anti pad4, by Abcam (1 mentions)
Rabbit anti mpo, by Cell Signaling Technology (1 mentions)
Anykd mini protean tgx gels, by Bio-Rad (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Nucleospin triprep kit, by Macherey-Nagel (1 mentions)
Rat anti mouse f4 80, by Bio-Rad (1 mentions)
Tunel staining kit, by Roche (1 mentions)
4 protocols using rabbit anti cith3
1
Quantification of Neutrophil Extracellular Trap Formation
2
Immunohistochemical Staining of Mouse Joints
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Frozen sections of mouse joints were thawed rapidly and then fixed in 2% paraformaldehyde/PBS (pH=7.4) for 12 min at room temperature. Sections were washed twice for 2 min in PBS. Sections were washed with PBS three times for 5 min each, followed by blocking with 10% normal goat serum overnight at 4 °C. Sections were probed with rabbit anti-MPO (Dako Denmark A0398) at a dilution of 1:500, rat anti-Ly6C/Ly6G antibody (BD Pharmingen 550327) at a dilution of 1:50 overnight at room temperature, rabbit anti-cit-H3 (Abcam Inc., ab5103) at a dilution of 1:500, monoclonal anti-DEK (BD Pharmigen 610948) 1:50, or with custom-made rabbit anti-DEK antibody6 (link) diluted in 10% normal goat serum at a dilutions of 1:50, 1:100 or 1:200 overnight at 4 °C. After washing in PBS for 5 min × 3, sections were incubated with secondary antibody; goat anti-rat IgG-AlexaFluor 594 (Invitrogen A-11007), at 1:200 for Ly6C/Ly6G or goat anti-rabbit IgG-AlexaFluor 594 (Invitrogen A-11037) at 1:200 for DEK and anti-cit-H3. Antibodies were diluted in 10% normal goat serum; sections were incubated for 45 min at room temperature.
3
Neutrophil Protein Isolation and Analysis
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Total protein was isolated by NucleoSpin TriPrep kit (Macherey-Nagel) from 5 × 106 neutrophils. All protein concentrations were determined with the MN Protein Quantification Assay (Macherey-Nagel). Western blotting was performed utilizing AnykD Mini-PROTEAN TGX Gels (Biorad) and nylon/nitrocellulose membranes (Biorad). Primary and secondary antibodies utilized were rabbit anti-MPO (Cell Signalling Technologies), rabbit anti-PAD4 (Abcam), rabbit anti-citH3 (Abcam), mouse anti-β-Actin (Sigma), anti-rabbit HRP (Santa Cruz), and anti-mouse HRP (Santa Cruz). HRP activity was detected by using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). Equal loading was verified using beta-actin. Western blots of citrullinated H3 (citH3) protein were prepared as described previously (34 (link)). Gel documentation, densitometric analysis, and protein quantification of the western blots was performed using the ChemiDoc XRS+ imaging system (Biorad) with the ImageLab 4.1 image analysis software (Biorad).
4
Histological Analysis of Thrombus Formation
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Thrombi were embedded in paraffin and 3 μm sections were deparaffinized and rehydrated as previously described34 (link). Sections were stained with H&E or prepared for immunohistochemistry. A 0.3% H2O2 was used for inhibition of endogenous peroxidase. Primary antibodies included rat anti-mouse Ly6b (neutrophils, AbD Serotec, Oxford, UK), rabbit anti-CitH3 (netting neutrophils, Abcam, Cambridge, UK), rabbit anti-mouse RIPK3 (Abcam, Cambridge, UK), anti-mouse MLKL (kindly provided by Andreas Linkermann, Dresden), and rat anti-mouse F4/80 (Serotec, Oxford, UK)35 (link). TUNEL staining kit (Roche, Mannheim, Germany) was used to detect dying cells inside the thrombus following the manufacturer’s description. Positive cells were quantified using the ImageJ software.
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