Anti il 10

The expression of both TNFα and IL10 proteins were detected in liver tissues of all groups using western blot assay. This assay was done as previously described^{66 (link)}. Briefly, liver specimens were lysed using RIPA lysis buffer. The obtained proteins were separated on SDS-PAGE gels and transferred to a polyvinylidene difluoride membrane (PVDF). PVDF membranes were separately incubated with the following mouse monoclonal primary antibodies: anti-TNFα (1:100 dilution, Santa Cruz Biotechnology, Cat # sc-52746), anti-IL10 (1:200 dilution, Santa Cruz Biotechnology, Cat # sc-365858), and anti-β-actin (housekeeping, 1:200 dilution, Cat # sc-47778). PVDF membranes were then incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:2000 dilution, Cell Signaling Technology, Beverly, MA, USA). The color was developed using tetramethylbenzidine (Sigma) and the band density was quantified by ImageJ software.

For the immunohistochemical evaluation, the following antibodies were used: anti-IL-4 (Santa Cruz Biotechnology, California, USA; 1:600), anti-IL-5 (Santa Cruz Biotechnology, California, USA; 1:100), anti-IL-10 (Santa Cruz Biotechnology, California, USA; 1:500), anti-IL-13 (Santa Cruz Biotechnology, California, USA; 1:700), anti-IL-17 (Santa Cruz Biotechnology, California, USA; 1:800), anti-CD4+ (Santa Cruz Biotechnology, California, USA; 1:25), anti-CD8+ (Santa Cruz Biotechnology, California, USA; 1:50), anti-MMP-9 (Santa Cruz Biotechnology, California, USA; 1:500) and anti-TIMP-1 (Santa Cruz Biotechnology, California, USA; 1:100).Immunohistochemistry was performed with the following sequence of procedures: antigenic recovery, endogenous peroxidase blockade and nonspecific binding blockade, incubation with the primary antibody, incubation with the secondary antibody and complex, counterstaining and assembly of the blades. The count was determined as described above for the evaluation of eosinophil density.

Chemicals were of the purest analytical grade. Human recombinants IL-2, IL-4, IL-6, IL-10, INF-γ, and LIF, calpain substrate [N-Suc-Leu-Tyr-AMC (7-amido-4-methyl-coumarin)], AA861 (specific inhibitor of 5-LOX), and E64D (specific inhibitor of calpain) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Mouse anti-cytochrome c antibody was from Cell Signalling Technology Inc. (Danvers, MA, USA); mouse anti-calpain-1 was from Calbiochem (Merck Darmstadt, Germany). Rabbit anti-LIF, anti-IL-2, anti-IL-4, anti-IL-6, anti-IL-10, anti-INF-γ, secondary antibodies conjugated to horseradish peroxidase (HRP), and enhanced chemiluminescence (ECL) kit were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Goat anti-rabbit conjugated to alkaline phosphatase (GAR-AP) was from Bio-Rad (Hercules, CA, USA).

The renal cortex was dissected from the right kidney of each mouse and the spleen, renal cortex, and renal medulla were homogenized at 4°C in RIPA buffer. Western blots were performed using splenic, renal cortical, and renal medullary homogenates as previously described (Mathis et al. 2012, 2013, 2014, 2014). Tumor necrosis factor (TNF)‐α and monocyte chemoattractant protein (MCP)‐1 were used as markers of inflammation, whereas interleukin (IL)‐10 was used as a marker of anti‐inflammation. TNF‐α was detected using a mouse monoclonal anti‐TNF‐α (1:250; Santa Cruz, Dallas, TX; 26 kDa). MCP‐1 was detected using a rabbit polyclonal anti‐MCP‐1 (1:2000; Abcam, Cambridge, MA; dimerized at ~37 kDa). IL‐10 was detecting using a mouse monoclonal anti‐IL10 (1:200; Santa Cruz, Dallas, TX; 20 kDa). Proteins were visualized using an HRP‐conjugated donkey anti‐mouse IgG (1:10000; Rockland, Limerick, PA), donkey anti‐rabbit IgG (1:1000; Rockland), or goat anti‐mouse (1:5000; Rockland), respectively. All Western blots were imaged and analyzed using the ChemiDoc MP Imaging System and ImageLab Software Version 5.1 (Bio‐Rad Laboratories, Inc., Hercules, CA). Western blot data are presented as a ratio of densitometry units of protein, based on band optical density, and normalized to total protein.

For immunohistochemistry analysis, all samples were previously submitted to deparaffinization. Endogenous peroxidase activity was blocked with H₂O₂ 3% three times during 10 minutes. Samples were then washed and blocked with bovine serum albumin 10% during 1 hour and then incubated with primary rabbit anti-mouse IgG antibodies: i) anti-NFκB (Santa Cruz, CA) at 1:500; ii) anti-NF-AT 1:500 (Santa Cruz, CA) and iii) anti-IL-10 (Santa Cruz, CA) at 1:500 during 2 hours at room temperature. Samples were washed twice with TBS- BSA 10% and then incubated with secondary antibody goat anti-Rabbit IgG at 1:1000 for 1 hour. After washing samples twice with BSA 2% diaminobenzidine (DAB) was added. Finally samples were washed again and counter stained with H&E. Slides were analyzed under light microscope on a blind fashion.