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3 protocols using il 10

1

Immunohistochemical Analysis of Immune Markers

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For immunohistochemical studies, the paraffin-embedded tissues were cut (sections of 4 μm), deparaffinized in xylene and rehydrated with alcohol. Antigen retrieval was performed by heating the tissue in the presence of citrate buffer [15 (link)]. Tissues were blocked for endogenous peroxidase with 3% hydrogen peroxidase in methanol and rinsed in Tris-HCl (pH 7.4). To reduce non-specific binding, sections were incubated for 30 min at room temperature. Samples were then incubated over-night at 4°C with anti-human antibodies that recognize CD4 (Spring Bioscence, CA, USA), CD8 (DAKOCytomation, CA, USA), CD20 (Biocare Medical, CA, USA), CD68 (Biocare Medical, CA, USA), IFNγ (Abbiotec, CA, USA), TNFα (Abbiotec, CA, USA), IL-10 (Abbiotec, CA, USA), RANTES/CCL5 (Santa Cruz Biotechnology, CA, USA) or TGF-β (Abbiotec, CA, USA). The next day, sections were incubated with rabbit anti-mouse IgG, a secondary antibody horseradish peroxidase (HRP) conjugate (Spring Bioscience, CA, USA), for 30 min at room temperature. For negative control of the immunohistochemical reactions, samples were incubated only with the secondary HRP-conjugated antibody. Reactions were revealed with diaminobenzidine (Dako, CA, USA) as a chromogen and the sections were counterstained in Meyer’s hematoxylin (Dako). Finally, samples were examined under a Nikon ECLIPSE E600 microscope.
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Comprehensive Immunohistochemistry Protocol for Kidney Tissue

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Immunohistochemistry staining was performed on formalin-fixed kidney tissues. Sections were deparaffinized with xylene and rehydrated in a graded alcohol series and then placed in PBS (pH 7.5). The microwave antigen retrieval procedure (citrate buffer, pH 6.0) was performed for 10 min. After that, sections were immersed in 3% hydrogen peroxide for 10 min to block endogenous peroxidase, then treated with 3% BSA (diluted in PBS) for blocking nonspecific binding sites, and incubated overnight at 4°C with the following primary antibodies: rabbit anti-mouse ki67 (1:2000, Abcam), IKKα (1:200, Abcam), NIK (1:200, Santa Cruz Biotechnology), p52 (1:200, Santa Cruz Biotechnology), RelB (1:300, Cell Signaling), IL10 (1:500, Abbiotec) and Foxp3+ (1:100, Santa Cruz Biotechnology). The next day, these slides were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-rat secondary antibody for 1 h at room temperature. 3,3-Diaminobenzidine tetrahydrochloride was applied to the slides for developing brown color. Counterstaining was carried out with Eosin. All slides contained duplicate sections, from which one served as a control for secondary antibody binding specificity. The positive areas were measured in five randomly chosen fields, and quantified blindly using an Olympus BX-URA2 camera.
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Immunohistochemical Detection of Inflammatory Markers

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For the detection of cytokines, inflammatory mediators and cell populations by immunohistochemistry, sections were treated as described before [21 (link)]. The slides were incubated overnight at 4°C with the primary antibodies that recognized HMGB1 (Abcam, UK; dilution 1:200), Caspase-3 (Abcam, UK; dilution 1:100), TGF-β (Abbiotec, CA, USA; dilution 1:300), MMP-9 (Santa Cruz Biotechnology, CA, USA; dilution 1:200), TNF-α (Santa Cruz Biotechnology, CA, USA; dilution 1:200) or IL-10 (Abbiotec, CA, USA; dilution 1:100) and prepared for visualization under a Nikon ECLIPSE E600 microscope.
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