A modified histoimmunochemistry-assisted method for in situ RPE evaluation

We have developed a method for morphological evaluation of retinal pigment epithelium (RPE) with minimal perturbation of RPE-Bruch’s membranechoriocapillaris complex (RBCC). To prepare RBCC, the anterior segments of the eye were removed. The remaining eye cup was radially cut into 4 to 6 pieces from periphery to the optic nerve head. Each piece was carefully dissected into 3 parts: sclera, RBCC and neurosensory retina. RBCC flatmount with RPE monolayer facing up was formed by several relaxing radial cuts. After immuno-staining with tissue specific markers, the RBCC could be distinguished as the superficial RPE monolayer and the underneath choriocapillaris layers by fluorescence microscopy. The density, distribution, and morphology of RPE cells varied among species. This method may have brought several advantages for RPE screening over other means, because of its straight forward approach, minimal manipulation of samples, plus there is no requirement for bleaching, which result in high efficiency for result readout. For a complete morphological study of RPE in situ, this method may be combined with other methods, such as cryosections, scanning electron microscopy, etc.