Figure 5

Hic-5 colocalizes with collagen I and with α-SMA in diseased glomeruli. (a) Double staining was undertaken to show colocalization of Hic-5, collagen I, and α-SMA within glomeruli from control, diabetic, and NTU281-treated diabetic kidneys. Because of the difficulty of using immunofluorescent techniques with paraffin-embedded tissues, light microscopy was used ( × 400 or × 1000 magnification), staining with diaminobenzadine (DAB, brown) and with vulcan red (VR, red). Typical glomeruli from animals at day 240 are shown. (b) Colorimetric analysis was undertaken to quantify the degree of colocalization of immunohistochemical staining. Color intensity in the red, green, and blue channels was analyzed on sections stained with Vulcan Red only or DAB only, and for double-stained sections. The patterns of red/green/blue color intensity produced could reliably be distinguished (*P<0.001). Examples of cells with single staining for each of Vulcan Red and DAB and double-stained cells are shown below their corresponding histogram. (c) A total of 20 randomly selected consecutive cells were analyzed for each condition of Hic-5+SMA, Hic-5+collagen I, and SMA+collagen I, according to these colorimetric criteria. In each case, colocalization was confirmed in at least 90% of cells.