*4.7. Immunofluorescence and Immunohistochemistry*

We performed immunostaining as previously described [7,41]. Immunofluorescence or immunohistochemistry was performed on 3-μm ice-cold acetone-fixed cryosections of kidneys using the following primary antibodies: anti-fibronectin, anti-CD4, anti-F4/80, anti-ICAM-1, anti-α-SMA (AbD Serotec, Oxford, UK). For indirect immunostaining, non-specific binding sites were blocked with 10% normal donkey serum for 30 min. Then, sections were incubated with the primary antibody for 1 h at room temperature or overnight at 4 °C. All incubations were performed in a humid chamber. For fluorescence visualization of bound primary antibodies, sections were further incubated with Cy3-conjugated secondary antibodies (Jackson Immuno Research, WG, USA) for 1 h in a humid chamber at room temperature. Slides were analyzed using a Zeiss Axioplan-2 imaging microscope with the computer program AxioVision 4.8 (Zeiss, Jena, Germany). For immunohistochemistry, after incubation with the primary antibody directed against α-SMA, biotinylated secondary antibody (Dako REAL™ EnVision™; Dako Denmark A/S, Glostrup, Denmark) was used. Immunohistochemical positive staining was consecutively revealed by the 3,3 - Diaminobenzidine Peroxidase Substrate Kit (Dako REAL™ EnVision™; Dako Denmark A/S, Glostrup, Denmark) in accordance with the manufacturer's instructions.

Quantitative analyses of infiltrating cells (CD4+ and F4/80+) and fibroblasts (α-SMA+) were counted in 15 non-overlapping, randomly chosen fields per kidney section under a 400× magnification. The average ratio of the fibronectin or ICAM-1-labeled area to the total area in the view (400×) was calculated using the software ImagJ (NIH, Bethesda, MD, USA). In addition, ICAM-1 expression was also analyzed using software ImagJ to calculate the mean gray value (integrated density to area).
