Figure 3

(A–D) Images showing the spatial path of a loop zone, in single (A–C) and whole-mount (D) views in confocal microscopy (frontal view, 6 µm section, immunofluorescence label with anti-CD34, green, and anti-collagen I, red, DAPI: blue). (E–M) Contacts between ECs from opposite walls of the loops (arrows). Note that the contacting ECs may have triangular (E), ovoid (F) or flattened (G) morphology in the histological sections and that planar contact may be extensive (H). Contacts can be symmetric (E) or asymmetric (I,J), in proximity (K) or not, and can acquire a peg-like aspect when ECs surround connective tissue (L). (M) Occasionally, smaller transcapillary cytoplasmic projections (antenna-like or filipodia-like) were seen. (N) Ultrastructural image of an endothelial projection (arrow) separating two luminal spaces (lu) of a loop. (O–R) Serial semithin sections showing an EC projecting intraluminally (in Figs. 4A,B, in which a perforated contact is shown, the region in O-R also appears in the image with a portion of the projecting cell -Fig. 4A- and with no presence of this projecting cell -Fig. 4B-). (S–Z) Single (T–Z) and whole-mounted (S) views in confocal microscopy (frontal view, 10 µm section and CD34 staining, DAPI: blue) showing the appearance and disappearance of endothelial contacts (arrowhead), including a peg-like structure (arrow). (E–M) Double-staining with anti-CD34 (brown) and anti-αSMA (red). (N) Ultrathin section. Uranyl acetate and Lead citrate. (O–R) Semithin sections stained with Toluidine Blue. Bar: A-D, 30 µm; (E–M,O–Z) 10 µm; (N) 8 µm.