Figure 4.

Type IV collagen (red) and CD31 immunoreactivities (rat clone MEC13.3 anti-mouse, green) viewed in two-dimensional projections of 1-μm confocal microscopic optical cross-sections of vessels in normal pancreatic islets (A), RIP-Tag2 tumors (B, C), MCa-IV breast carcinomas (D–F), and Lewis lung carcinoma (G). Physical section thickness, 80 μm. Type IV collagen immunoreactivity completely envelops most vessels. Arrows mark regions of type IV collagen immunoreactivity that extend beyond the CD31-immunoreactive perimeter of tumor vessels. Arrowheads mark tiny defects (0.3 to 2.5 μm in diameter) in type IV collagen immunoreactivity and/or CD31 immunoreactivity of vessels in the three types of tumor, as detailed in Table 1 ▶ . There are several reasons why CD31 immunoreactivity of endothelial cells appears as a single band instead of two discrete (luminal and abluminal) plasma membranes in these images. To visualize the immunofluorescence in 1-μm optical sections of endothelial cells, the confocal signal was amplified to a point plasma membranes appear wider than they really are. The apparent widening of the membranes is exaggerated by slight tilting from vertical of vessels not cut precisely in cross-section. The separation of the two membranes is less than the thickness of the optical sections (except in the region of the nucleus) and is of the same order of magnitude as the resolution of the ×40 objective lens. Because of the interaction of these factors, the width of the signal from both membranes blurs the space between them, and they fuse into one fluorescent band. Scale bars: 5 μm (A, B, G); 7.5 μm (C); 45 μm (D–F).