[Frontiers in Bioscience E4, 1571-1581, January 1, 2012]

A modified histoimmunochemistry-assisted method for in situ RPE evaluation

Jingfa Zhang1,4, Guoxu Xu2, Limei Zhang4, Limin Gu4, Hua Xu4, Lixia Lu1,4, Juan Wang1,4, Furong Gao1,4, Jing-Ying Xu1,4, Fang Wang1, Weiye Li1,3,4, Guo-Tong Xu1,4

1Department of Ophthalmology, Shanghai Tenth People's Hospital, and Tongji Eye Institute, Tongji University School of Medicine, Shanghai, China, 2Department of Ophthalmology, Second Affiliated Hospital of Soochow University, Suzhou, China, 3Department of Ophthalmology, Drexel University College of Medicine, Philadelphia, PA, USA, 4Stem Cell Research Center and Department of Regenerative Medicine, Tongji University School of Medicine, Shanghai, China

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods
3.1. Reagents
3.2. Experimental animals and sodium iodate (SI)-induced retinal pigment epithelium (RPE) degeneration rat model
3.3. RPE-Bruch's membrane-choriocapillaris complex (RBCC) isolation and immunohistochemistry
4. Results
4.1. The procedure for RBCC isolation and observation
4.2. The morphology and distribution of the microvilli
4.3. Pigmented RPE cells can be detected without bleaching
4.4 .Morphology of RPE cells varies with species
4.5 .The study of RBCC in animal models
5. Discussion
6. Acknowledgement
7. References

1. ABSTRACT

We have developed a method for morphological evaluation of retinal pigment epithelium (RPE) with minimal perturbation of RPE-Bruch's membrane-choriocapillaris 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.