Urve method [10]. Total RNA was extracted from snap-frozen kidney tissues with TRIzol Reagent (Invitrogen). Total RNA were reverse-transcribed and amplified in triplicate using IQ SYBR green supermix reagent (Bio-Rad, Herculus, CA) with a real-time PCR machine (Bio-Rad, Herculus, CA), Nafarelin site according to the manufacturer’s instructions. The specificity of real-time PCR was confirmed by melting-curve analysis. The expression levels of the target genes were normalized to the GAPDH level in each sample. The following are the primer sequences: IL-6: Forward 59- GAGGATACCACTCCCAACAGACC-39 and reverse 59- AAGTGCATCATCGTTGTTCATAC-39; TGF-b1: Forward 59- CAACAATTCCTGGCGTTACCTTGG-39 and reverse 59GAAAGCCCTGTATTCCGTCTCCTT-39; CXCL16: Forward 59- ACCCTTGTCTCTTGCGTTCTTCCT-39 and reverse 59ATGTGATCCAAAGTACCCTGCGGT-39; IL-4: Forward 59ATCGGCATTTTGAACGAGGTC-39 and reverse 59- GAGGACGTTTGGCACATCCA-39; IL-13: Forward 59CAGCCTCCCCGATACCAAAAT-39 and reverse 59GCGAAACAGTTGCTTTGTGTAG-39; GAPDH: Forward 59TGCTGAGTATGTCGTGGAGTCTA-39 and reverse 59AGTGGGAGTTGCTGTTGAAATC-39.ImmunohistochemistryImmunohistochemical staining was performed on paraffin sections. Antigen retrieval was performed with antigen unmasking solution (Vector Laboratories, Burlingame, CA). Endogenous peroxidase activity was quenched with 3 H2O2. After blocking, slides were incubated with primary (-)-Calyculin A web antibody in a humidified chamber overnight. After washing, slides were incubated with appropriate secondary antibody and ABC solution sequentially according to the Vectastain ELITE ABC kit (Vector Laboratories, Burlingame, CA). The reaction was visualized by incubation with DAB solution for an appropriate period of time. Slides were then counterstained with hematoxylin, dehydrated, and coverslipped. The images were acquired and analyzed by NIS Element software with Nikon microscope image system.Figure 1. IL-6 is induced in the kidney following obstructive injury. A. Graphic presentation shows IL-6 mRNA induction. ** P,0.01 vs normal control kidney. n = 4. B. Representative photomicrographs of kidney sections stained for IL-6 (brown) and counter stained with hematoxylin (blue) (Original magnification: X400). doi:10.1371/journal.pone.0052415.gImmunofluorescenceRenal tissues were embedded in OCT compound, snap-frozen on dry ice, cut at 5 mm thickness using a cryostat, and mounted on Superfrost Plus microscope slides. After fixation, nonspecific binding was blocked with serum-free protein block (DAKO). Slides were then incubated with goat anti-MCP-1 antibody (R D Systems) followed by Alexa-488 conjugated donkey anti-goat antibody (Invitrogen), rabbit anti-collagen I antibody (Rockland) followed by Alexa-488 conjugated donkey anti-rabbit antibody (Invitrogen), rabbit anti-fibronectin antibody (Sigma) followed by Alexa-488 conjugated donkey anti-rabbit antibody (Invitrogen), or rabbit anti-a-SMA antibody (Abcam) followed by Alexa-488 conjugated donkey anti-rabbit antibody (Invitrogen). For double immunofluorescence, kidney sections were fixed and stained with primary antibodies followed by appropriate secondary antibodies sequentially. Slides were mounted with mounting medium with DAPI. Fluorescent intensity was visualized using a microscope equipped with a digital camera (Nikon, Melville, NY). Quantitative evaluation of sections stained with antibodies to a-SMA,of C57BL/6J were purchased from the Jackson Laboratory. Male WT or IL-6 KO mice at 8?0 weeks old age, weighing 20?0 g were anesthetized by i.p. injection.Urve method [10]. Total RNA was extracted from snap-frozen kidney tissues with TRIzol Reagent (Invitrogen). Total RNA were reverse-transcribed and amplified in triplicate using IQ SYBR green supermix reagent (Bio-Rad, Herculus, CA) with a real-time PCR machine (Bio-Rad, Herculus, CA), according to the manufacturer’s instructions. The specificity of real-time PCR was confirmed by melting-curve analysis. The expression levels of the target genes were normalized to the GAPDH level in each sample. The following are the primer sequences: IL-6: Forward 59- GAGGATACCACTCCCAACAGACC-39 and reverse 59- AAGTGCATCATCGTTGTTCATAC-39; TGF-b1: Forward 59- CAACAATTCCTGGCGTTACCTTGG-39 and reverse 59GAAAGCCCTGTATTCCGTCTCCTT-39; CXCL16: Forward 59- ACCCTTGTCTCTTGCGTTCTTCCT-39 and reverse 59ATGTGATCCAAAGTACCCTGCGGT-39; IL-4: Forward 59ATCGGCATTTTGAACGAGGTC-39 and reverse 59- GAGGACGTTTGGCACATCCA-39; IL-13: Forward 59CAGCCTCCCCGATACCAAAAT-39 and reverse 59GCGAAACAGTTGCTTTGTGTAG-39; GAPDH: Forward 59TGCTGAGTATGTCGTGGAGTCTA-39 and reverse 59AGTGGGAGTTGCTGTTGAAATC-39.ImmunohistochemistryImmunohistochemical staining was performed on paraffin sections. Antigen retrieval was performed with antigen unmasking solution (Vector Laboratories, Burlingame, CA). Endogenous peroxidase activity was quenched with 3 H2O2. After blocking, slides were incubated with primary antibody in a humidified chamber overnight. After washing, slides were incubated with appropriate secondary antibody and ABC solution sequentially according to the Vectastain ELITE ABC kit (Vector Laboratories, Burlingame, CA). The reaction was visualized by incubation with DAB solution for an appropriate period of time. Slides were then counterstained with hematoxylin, dehydrated, and coverslipped. The images were acquired and analyzed by NIS Element software with Nikon microscope image system.Figure 1. IL-6 is induced in the kidney following obstructive injury. A. Graphic presentation shows IL-6 mRNA induction. ** P,0.01 vs normal control kidney. n = 4. B. Representative photomicrographs of kidney sections stained for IL-6 (brown) and counter stained with hematoxylin (blue) (Original magnification: X400). doi:10.1371/journal.pone.0052415.gImmunofluorescenceRenal tissues were embedded in OCT compound, snap-frozen on dry ice, cut at 5 mm thickness using a cryostat, and mounted on Superfrost Plus microscope slides. After fixation, nonspecific binding was blocked with serum-free protein block (DAKO). Slides were then incubated with goat anti-MCP-1 antibody (R D Systems) followed by Alexa-488 conjugated donkey anti-goat antibody (Invitrogen), rabbit anti-collagen I antibody (Rockland) followed by Alexa-488 conjugated donkey anti-rabbit antibody (Invitrogen), rabbit anti-fibronectin antibody (Sigma) followed by Alexa-488 conjugated donkey anti-rabbit antibody (Invitrogen), or rabbit anti-a-SMA antibody (Abcam) followed by Alexa-488 conjugated donkey anti-rabbit antibody (Invitrogen). For double immunofluorescence, kidney sections were fixed and stained with primary antibodies followed by appropriate secondary antibodies sequentially. Slides were mounted with mounting medium with DAPI. Fluorescent intensity was visualized using a microscope equipped with a digital camera (Nikon, Melville, NY). Quantitative evaluation of sections stained with antibodies to a-SMA,of C57BL/6J were purchased from the Jackson Laboratory. Male WT or IL-6 KO mice at 8?0 weeks old age, weighing 20?0 g were anesthetized by i.p. injection.
