Formalin-fixed and paraffin-embedded kidney sections were blocked with 5% fetal bovine serum, and then the MPO-stained with rabbit anti-myeloperoxidase antibody (Abcam, catalog ab208670) in combination with a second Cy3-conjugated goat anti-rabbit IgG (H + L) antibody (Servicebio, catalog GB21303). The chromatin was stained with DAPI (Thermo Fisher Scientific). Colocalization of myeloperoxidase and chromatin was recognized as NETs. Ten glomeruli per animal were analyzed for NET staining and the percentage of NET-stained glomeruli calculated.
Rabbit anti myeloperoxidase antibody
Manufactured by Abcam
Sourced in Germany
Rabbit anti-myeloperoxidase antibody is a primary antibody that specifically binds to the myeloperoxidase protein. Myeloperoxidase is an enzyme expressed in neutrophils and monocytes, and is involved in the production of hypochlorous acid, a potent antimicrobial agent.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Eclipse ti u 100, by Nikon (1 mentions)
Rabbit anti histone h3 citrulline r2 r8 r17 antibody, by Abcam (1 mentions)
Nis elements br 3, by Nikon (1 mentions)
Dako fluorescent mounting medium, by Agilent Technologies (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
Dako envision system hrp dab, by Agilent Technologies (1 mentions)
Nanozoomer digital pathology scanner, by Hamamatsu Photonics (1 mentions)
4 protocols using rabbit anti myeloperoxidase antibody
1
Immunohistochemical Analysis of NETs in Kidney Sections
2
Induction and Visualization of NETosis
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To induce NETosis, freshly isolated bone marrow-derived neutrophils (1.5 × 105) were seeded on poly-L-lysine coated coverslips. Cells were stimulated with 4 μM ionomycin for 3 h. After that, cells were fixed with 4% PFA, blocked and incubated with a polyclonal rabbit anti-myeloperoxidase antibody (1:100, Abcam), or alternatively, with a rabbit anti-histone H3 (citrulline R2+R8+R17) antibody (1:500, Abcam). Cells were further incubated with a secondary goat anti-rabbit Alexa Fluor 488-conjugated antibody (1:1,000, Cell Signaling Technology), counterstained with DAPI and mounted in Dako fluorescent mounting medium (Dako). NETs were finally visualized using an inverted microscope (Eclipse Ti-U 100, Nikon) and the NIS Elements BR 3.10 software package.
3
Lung Tissue Analysis: Quantifying MPO and RAGE
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Paraffin embedded lungs were sectioned into 5-micron slices and mounted on slides. Serial tissue sections were stained for hematoxylin and eosin (H&E) and for myeloperoxidase (MPO) (rabbit anti-myeloperoxidase antibody, 1:8000, abcam) and for RAGE (human anti-RAGE antibody, 50 µg/ml). Secondary staining was performed with HRP-conjugated goat anti-rabbit or goat anti-human antibody (1:200). Staining for MPO and for RAGE (brown staining) was quantified by IHC on five sections from each lung section using Image Pro Plus software (Media Cybernetics, Silver Spring, MD).
4
Quantification of Kidney and Liver Oxidative Stress
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Briefly, 4 µm sections of kidney and liver samples were obtained. For nitrotyrosine, the sections were blocked with PBS / 1% BSA (PBA) for 1 h and then incubated with 2% goat serum in PBA for 30 min. For myeloperoxidase, the sections were blocked with 10% goat serum for 15 min.
The slides were then incubated with rabbit anti-nitrotyrosine antibody (1:2000; Merck Millipore, Darmstadt, Germany) or with rabbit anti-myeloperoxidase antibody (1:25; Cat# ab9535, Abcam, Cambridge, UK) and then incubated with labelled polymer-HRP antibody (Dako EnVision+ System-HRP-DAB, K4010). DAB chromogen solution was used and a counter-staining was performed with Harris haematoxylin. Images were acquired using NanoZoomer Digital Pathology Scanner (Hamamatsu Photonics K.K., Japan) and analysed using the NDP Viewer software. The relative quantification of nitrotyrosine immunostaining was achieved through densitometry analysis using NIH ImageJ 1.36 imaging software (NIH, Bethesda, MD, USA) and it is expressed as arbitrary units.
The number of MPO positive cells was counted in 10 randomly selected fields (x 400) in a doubleblinded manner.
The slides were then incubated with rabbit anti-nitrotyrosine antibody (1:2000; Merck Millipore, Darmstadt, Germany) or with rabbit anti-myeloperoxidase antibody (1:25; Cat# ab9535, Abcam, Cambridge, UK) and then incubated with labelled polymer-HRP antibody (Dako EnVision+ System-HRP-DAB, K4010). DAB chromogen solution was used and a counter-staining was performed with Harris haematoxylin. Images were acquired using NanoZoomer Digital Pathology Scanner (Hamamatsu Photonics K.K., Japan) and analysed using the NDP Viewer software. The relative quantification of nitrotyrosine immunostaining was achieved through densitometry analysis using NIH ImageJ 1.36 imaging software (NIH, Bethesda, MD, USA) and it is expressed as arbitrary units.
The number of MPO positive cells was counted in 10 randomly selected fields (x 400) in a doubleblinded manner.
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