Related isotype immunoglobulins

Immunofluorescence and immunohistochemical stains were performed on formalin-fixed, paraffin-embedded 4-µm sections. The sections were rehydrated, and antigens were retrieved using heated citrate. For immunohistochemistry, staining was visualized using horseradish peroxidasecoupled secondary antibodies (Vectastain Elite; Vector Laboratories, Peterborough, UK). For immunofluorescence, the slides were exposed to FITC-(1:200) or rhodaminelabeled (1:500) secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA). The sections were mounted in Vectashield medium that contained DAPI (Invitrogen, Carlsbad, CA, USA). Images were acquired using a fluorescence microscope (Nikon, Tokyo, Japan). Related isotype immunoglobulins (Jackson ImmunoResearch) were used as negative controls for all stainings. All immunohistochemical and immunofluorescence analyses were repeated at least three times, and representative images are presented.

Cellular Physiology and Biochemistry visualized using horseradish-peroxidase coupled secondary antibodies (Vectastain elite, Vector Labs). Related isotype immunoglobulins (Jackson ImmunoResearch, USA) were used as the negative controls. All immunohistochemical analyses were repeated at least three times and representative images were presented. All analyses were performed in a masked manner. The quantitation of immunostaining was carried out on coded slides. The expression of collagen I, fibronectin, MCP-1, ICAM-1, PAI-1, and TGF-β1 in the entire cortex (a cross-section of the kidney) was determined using an image analyzer version 6.1 (WinRoof) and the data were expressed as a percentage of the positive area as previously described [17] [18] [19] . The number of F4/80+ cells in the tubulointerstitium was counted in 20 consecutive fields under ×400 magnification by means of a 0.0625-mm 2 graticule fitted in the eyepiece of the microscope and expressed as cells per mm 2 .