Anti mpo

Cells or NET-precipitated proteins were lysed on ice in SDS lysis buffer (2% SDS, 62.5 mM Tris at pH 6.8, 5% 2-mercaptoethanol, 10% glycerol) supplemented with Complete Roche Inhibitor Cocktail (Complete tablets Mini Easypack [catalog 04693124001] and PhosSTOP Easypack [catalog 04906837001]) and centrifuged at 13,000g for 15 minutes at 4°C. Whole-cell lysates (20–30 μg protein) were subjected to SDS-PAGE electrophoresis on 12.5% gels and then transferred to an Immobilon-PSQmembrane (catalog SEQ85R; Millipore). Membranes were blocked with 5% skimmed milk in TBS plus Tween 20 and then incubated with anti-MPO (1:3000 dilution; Dako) as loading control (78 (link)) or anti–IL-33 (1:1000 dilution) specific antibodies (mouse “Nessy” antibody; R&D). For HL-60 protein extracts, mouse anti–human tubulin antibody was used as loading control (1:3000 dilution, catalog A11126; Thermo Fisher Scientific). Detection was performed using relevant HRP-linked antibodies (anti–goat-HRP, anti–rabbit-HRP, anti–mouse-HRP; catalog AP180P, 12-348, and 12-349, respectively; all purchased from Millipore) and enhanced chemiluminescent-detection reagents (Amersham Biosciences, RPN2109). Unedited gels are provided in the online supplement.

For antigen retrieval, paraffin sections were digested by the same method with ISEL for MPO, or using 10 mM sodium citrate buffer for active caspase-3, sFGL2 and FcγRIIB. The sections were then blocked by peroxidase-blocking reagent and labelled by anti-MPO (1:600 dilution, DAKO), anti-active caspase-3 antibody (1:100 dilution, R&D System, Minnesota, USA), anti-sFGL2 (1:200 dilution, Abcam, Cambridge, UK) or anti-FcγRIIB (1:200 dilution, Abcam) antibodies at 4°C overnight. The antibody binding was revealed by AEC for MPO and active caspase-3, and DAB for sFGL2 and FcγRIIB. MPO + cells and active caspase-3+ cells in the renal cortex were manually counted, while the expression of sFGL2 and FcγRIIB were semi-quantitatively scored using optical volume density (OD × mm²) analysis (Image-Pro Plus 6.0, Media Cybernetics Inc., Bethesda, USA) in 20 fields at 400× magnification.

A NET-remnants ELISA, detecting complexes of DNA and myeloperoxidase (MPO) both found in NETs, was run for all samples (cohort 1-3) as previously described (29 (link)). NET-remnants will be referred to as NETs for the rest of the article. Briefly, the plate was coated at 4°C overnight with anti-MPO (DAKO, Carpinteria, CA, USA) followed by washing (0,9% NaCl and 0,05% Tween 20 in deionized H₂O) and addition of a peroxidase-labelled anti-DNA antibody (detection antibody of the Human Cell Death Detection ELISAPLUS; Roche Diagnostics GmbH, Mannheim, Germany) together with serum sample. After 2 hours of incubation the plate was washed, ABTS-substrate (Roche Diagnostics) added and after 40 min at 37°C the plate was read at 405 nm in a VersaMax ELISA microplate reader (Molecular Devices, Sunnyvale, CA, USA). All samples were run in duplicates, a 9-point standard curve with NETs produced in the lab (29 (link)) was included on each plate as well as positive control and blank. Values were interpolated from the standard curve and expressed as arbitrary units. Samples below detection limit were given the value 0.

For immunohistochemistry (IHC) staining, the following antibodies were applied on formalin-fixed, paraffin-embedded tissue using a routine immunoperoxidase technique [15 , 16 ] and experimental sequential IHC [17 (link)]: anti-CD3 (Dako M7254), anti-CD4 (Dako M7319), anti-CD8 (Dako M7103), anti-CD20 (Dako M0755), anti-Bcl2 (DakoM0887), anti-MPO (Dako C7246), anti-CD61 (Dako C7280). Two chromogens were used, DAB (DAB chromogen) as fix and NovaRed (VECTOR NovaRed) as removable chromogen for sequential IHC [17 (link)].

Full-thickness small intestine samples were fixed in buffered formalin (4% w/v formaldehyde and acetate buffer 0.05 M) for 24–48 h and routinely embedded in paraffin. Three-micrometer-thick sections were stained with haematoxylin–eosin (HE) for morphologic evaluation. Additional sections were mounted on poly-L-lysine-coated slides for immunohistochemical analysis of neutrophil infiltration. Immunohistochemistry was performed with the avidin–biotin–peroxidase method^{5 (link)} using a polyclonal antibody anti-MPO (DAKO, Glostrup, Denmark). Endogenous peroxidase activity was blocked by immersing sections for 10 min in a solution of 3% hydrogen peroxide in water and the primary antibody was incubated overnight at 4 °C. Specific biotinylated secondary antibody and avidin–biotin–peroxidase complex were consecutively applied, each for 1 h at room temperature. The immunohistochemical reaction was developed with diaminobenzidine–hydrogen peroxide reaction. Sections were counterstained with haematoxylin. Neutrophil infiltration was evaluated only in whole well oriented sections of intestine, counting MPO⁺ cells in four high power fields (400x, diameter 0.55 mm). MPO value has been reported as the average of MPO⁺ cells for field in each layer.