Supplementary Files

Files in this Data Supplement:

• Supplementary Figure S1 - 5.21 MB (.tif)
  Rupture of Bruch's Membrane. A single lesion (1-day post laser) was imaged in a flatmount orientation, then embedded and cryosectioned to show the lesion in cross section. (A) A projection of a confocal z-series shows the distribution of fragmented nuclei (blue), disrupted RPE cells (green), and occasional isolectin-positive cells (red) in this flat-mount preparation. An autofluorescent circular defect with irregular borders was detected deep to the RPE, corresponding to ruptured Bruch's membrane (arrowhead). (B) A confocal single plane image collected beneath the RPE, showing the autofluorescent ring defect in Bruch's membrane. Side views (xz and yz) show the ring defect in cross section (arrowheads). (C) Arrows show the location of the defect in Bruch's membrane in this cryosection. An extended area of coagulative necrosis with ghost-like choroidal vessels (arrowhead) was observed below the defect. Scale bar, 100 mm.

• Movie 1 - 13.9 MB (.avi)
  Rat choroid/RPE flat mount 4 days after laser: Movie from confocal Z-series taken at 0.4884 mm intervals. The movie begins at the top of the lesion (subretinal), ending toward the choroid. In the center, vascular tubes are visualized with isolectin IB₄ (red) coming from the deep choroidal layers. To the lower right, microglia cells (also isolectin IB₄ positive) are seen in the superficial layers, disappearing in the deeper ones. Phalloidin (green) shows the morphology of both, normal and proliferating RPE cells. DAPI (blue) stains the nuclei.

• Movie 2 - 10.9 MB (.avi)
  Rat choroid/RPE flat mount 14 days after laser: This movie was collected as described in Movie 1. Isolectin labeled (red) vascular tubes are visible near the top of the lesion. Choriocapillaris is visible in the upper left corner. Deeper in the lesion, a fully formed vascular complex joins a network of large feeder vessels. Phalloidin-labeled RPE cells (green). DAPI stains the nuclei (blue).

• Movie 3 - 15 MB (.avi)
  Isolectin IB₄ / CD11b Co-localization in rat choroid/RPE flat mount 7 days after laser: This movie begins at the bottom of the lesion (choroid) and ends at the top (subretinal). In order to visualize the location of microglia relative to new vessels, they were labeled with CD11b antibody (green) and vessels with isolectin IB₄ (red). Microglia were most abundant on top of the lesion, but were also present around the vessels. DAPI stained nuclei (blue).