Double Immunohistochemistry for Vascular and Renal Analysis

Double immunohistochemical staining with mouse anti-CD34 (Dako, Glostrup, Denmark) and anti-SMA (Dako) was performed to identify endothelial cells and smooth muscle cells of the arterial media (Figure 1A). The PAS stain was then applied to the sections to identify the tubular basement membranes, Bowman’s capsules, sclerotic glomeruli, and interstitial fibrosis (Figure 2F,G, Figure 3A,G, Figure 4F,G, Figure 5C,F,G and Figure 6G). Details of the tissue preparation are described elsewhere [7 (link),8 (link),9 (link),10 (link)]. Each stained specimen was digitized using virtual slide microscopy (NanoZoomer RS™; Hamamatsu Photonics, Hamamatsu, Japan), using a 40× optical lens.