Digital camera

Monocyte adhesion to the wall of the thoracic aorta in rats was examined by NEMOes as described previously [15 (link), 16 (link)]. Briefly, rats were sacrificed and perfused with normal saline followed by 10% buffered formalin into aorta. After fixation, the aorta was divided into segments 8–12 mm long. Each segment was then placed in 0.05% hydrogen peroxidase, then incubated with mouse anti-rat CD68 antibody (Serotec, Raleigh, NC, USA), and diluted 1 : 100 in phosphate-buffered saline (PBS). After the staining, the segments were cut open longitudinally along the ventral side with scissors. Specimens were viewed under a microscope (E800; Nikon, Tokyo, Japan) connected to an XYZ controller and a digital camera (Media Cybernetics Inc., Silver Spring, MD, USA). Pictures were taken at various focal lengths with an automatically regulated Z-stepper and the clearest images were selected automatically to produce a composite image of the whole thoracic aorta by Image-Pro4.5 J (Planetron, Tokyo, Japan) [16 (link)]. To quantitate the exact number of monocytes adhering to the endothelium, we counted separately the numbers of CD68-positive tear-shaped cells around the openings of intracostal arteries in each aorta (1400 μm × 1000 μm) (Figure 1(b)). The cell density in each area was calculated as the cell count divided by the total area by examiners blinded to the treatment regimen.