Figure 5.

BM perforation dilation is a transient event. (A) Quantification of perforation number on initial lymphatic vessels in crawl-in assays at time points t = 1.5 h and t = 24 h. Six different tissues (up to two areas per tissue) were imaged at the indicated time points and analyzed morphometrically. Perforation number and area at t = 0 h was set to 100%. Each dot represents one analyzed area. Error bars show mean ± SEM (B) Quantification of perforation area on initial lymphatic vessels in crawl-in assays at time points t = 0 h, t = 1.5 h, and t = 24 h. Six different tissues (up to two areas per tissue) were imaged at the indicated time points and analyzed morphometrically. (C) Initial lymphatic vessel (BM staining for collagen IV; blood capillary also visible in bottom right corner) visualized using a spinning disk confocal microscope (maximum projection of z-stack, 4 planes, 0.5 µm distance). Bar, 20 µm. (D) Enlargement of inset in C showing perforation size at t = 0 h, t = 1.5 h, and after 24 h of addition of DCs to ear explant. Bar, 10 µm. (E) Representative analysis of the perforation area after migration of DCs into the lumen of an initial lymphatic vessel. During the course of 24 h, BM perforations get dilated (1.5 h) and relax again (24 h). Enlargement of inset 1–3 in D showing perforation size at t = 0 h, t = 1.5 h, and after 24 h. Numbers below each image denote perforation area (red line) in square micrometers. Bars, 2 µm. Images are representatives of one of at least three independently performed crawl-in assays.