Merged confocal image of mouse (C57BL/6J, postnatal day 60) retinal pigment epithelium (RPE) flatmount. The image is stained for ZO-1 (Green, tight junctions between cells) and nucleus (Red, Propidium Iodide). The RPE cells are in general regularly shaped and arranged polygonal cells, however, their shape and arrangement changes significantly according to their location and age.
Mouse eye was enucleated, fixed for 10 min in 10% neutral buffered formalin, and washed three times with PBS. Flatmounting was done by making four radial cuts from the center of the cornea back toward the optic nerve. The iris and retina were removed using forceps and additional incisions were made at the ciliary body/cornea margin to relieve tension from the sclera. The RPE flatmounts were stained for rabbit anti-ZO1 antibody (1:100 dilution, catalog no. 61-7300; Invitrogen, Carlsbad, CA) and goat anti-rabbit IgG secondary antibody (catalog no. O11038; Invitrogen). Imaging of the flatmounts was performed with a confocal imaging system (model C1; Nikon) using argon laser excitation at 488 nm. Confocal images were digitally merged (Adobe Photoshop CS2; Adobe, San Jose, CA).