Figure 1. SC visualized in 3D using whole mounts.

(A) Enface view of adult SC and other limbal vessels. (Left) DIC image of a whole mount prepared as in Figure S1. SC and limbal vessels are located in the limbus, just inside the dark pigmented band (see Figure S1). (Middle) Localization of the limbal vessels stained with endomucin in a Z-projection of confocal limbal stacks encompassing the entire whole mount. The whole-mount stacks were Z-depth color-coded (ICE LUT, see Figure S1). Color scale shows depth code colors of all structures stained with endomucin from inside (in) the eye to outside (out), with the SC being the most internal coded structure. (Right) Overlay of DIC and immunofluorescence image. (B) Higher magnification showing SC in relation to blood vessels of the LVP. BV, blood vessel; cc, collector channel. The blood vessels (magenta) are closer to the external ocular surface than SC (cyan). (C, D) Imaged whole mounts with the basement membrane marker collagen IV (COLIV) used to highlight SC in enface (XY) and conventional (XZ) orientations. (C) XY view; the icon in the lower left corner indicates the orientation of SC (also used in subsequent figures). The inner wall (IW) is closer to the reader, and the outer wall (OW) is away from the reader. (D) XZ view through the plane indicated by yellow lines in (C). In this XZ orientation, the lumen of SC (*) is evident between the COLIV-labeled inner and outer walls (compare to E). (E) Frozen section with COLIV labeling overlaid on its DIC image. The similarity between the XZ-represented whole mount and frozen section is clear. Note the characteristic bulbous undulations of the inner wall protruding into the lumen (arrowheads). Scale bar, (A) 500 µm, (B) 100 µm, and (C–E) 20 µm.