Kinetics of heterophil influx into collagen CAM onplants. Samples of normal CAM from embryos without onplants (NCAM) and collagen onplants supplemented with angiogenic growth factors (bFGF/VEGF) or buffer alone (control) were excised with the underlying CAM, embedded in OCT compound, and frozen. Cryosections were immunohistochemically stained with chMMP-9 antibody and counterstained with Mayer hematoxylin. Digital images were collected at × 10, × 20, and × 40 magnifications and analyzed. (A) Representative sections of control (left panel) and growth factor–supplemented (right panel) collagen onplants harvested at 72 hours (original magnification × 10). Arrows point to dark brown–stained cells and cell clusters confirmed to be chMMP-9–positive heterophils. (B) At an original magnification of × 40, morphologic features of chMMP-9–positive cells are consistent with the characteristics of heterophils (ie, multilobed nuclei [bluish] and granular cytoplasm [brownish]). (C) Kinetics of heterophil influx into collagen CAM onplants was determined over the 72-hour time course. The original × 20 magnification images of normal CAM (NCAM), control, and growth factor–containing (bFGF/VFGF) onplants were overlaid with a 9 × 7 square grid and analyzed (data from 9 to 39 images per time point from 2 independent experiments). chMMP-9–positive heterophils were counted in the squares occupied with tissue. Data are presented as the mean ± SEM from the numbers of chMMP-9–positive cells per square. ^*P < .001, determined in the 2-tailed Student t test.