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Lixia Lu, Jingfa Zhang, Weiye Li, Guo-Tong Xu; A Histoimmunochemistry-assisted Method For Evaluation Of Retinal Pigment Epithelium (RPE) Within An Intact RPE-Bruch’s Membrane-choriocapillaris Complex. Invest. Ophthalmol. Vis. Sci. 2011;52(14):932.
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To develop a new method for comprehensive morphological evaluation of retinal pigment epithelium (RPE) with minimal perturbation of RPE-Bruch’s membrane-choriocapillaris complex (RBCC).
The eyes of mice, rats, rabbits, pig and monkey were obtained and fixed in PBS buffered 4% paraformaldehyde solution. For RBCC preparation, the anterior segments of the eye were gently removed under the dissecting microscopy. The remained eye cup was radially cut into 4 to 6 pieces from periphery to the optic nerve head. Each piece can be carefully dissected into 3 parts: sclera, retina and RBCC. And then, RBCC was immuno-stained for evaluation of the RPE cells and choriocapillaris. Before examination under fluorescence microscopy, RBCC flatmount with RPE monolayer facing up was prepared by several relaxing cuts, depending on the size of the RBCC.
After staining with tissue specific markers, RPE 65 antibody for RPE and lsolectin for choricapillaris endothelial cells, the RBCC could be distinguished as the superficial RPE monolayer and the underneath choriocapillaris layers. The RPE cells appeared polygonal with visible nucleuses and prominent nucleolus under fluorescence microscopy. The density, size and distribution of RPE nucleuses varied among species. The detachment of RPE cells from Bruch’s membrane can also be observed.
This method may have several advantages to screen the changes of RPE than other means, because of its relative simplicity, minimal manipulation of samples, no requirement for bleaching, en bloc examination, and high efficiency for result readout. For a completed morphological study of RPE, this method may be combined with other methods, such as cryosections, scanning electron microscopy, etc.
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