A Simple and Rapid Method for Simultaneous Isolation of Mouse Retina and RPE Wholemounts.

PURPOSE: Retina and retinal pigment epithelium (RPE) wholemounts are important models for studying the pathophysiology of retinal-related ophthalmic diseases, such as age-related macular degeneration and diabetic retinopathy. Currently, there is no method available for simultaneously obtaining retina and RPE wholemounts. The aim of this study is to develop a simple, rapid, and effective technique for the simultaneous isolation of mouse retina and RPE wholemounts.

METHODS

We developed a novel, streamlined procedure for the efficient isolation of intact retina and RPE wholemounts from mouse eyes. The method involves minimal dissection and uses basic laboratory equipment, allowing the entire process to be completed in approximately 2 to 5 minutes per sample. The study also explores the impact of different fixation times on the structural integrity and quality of both retina and RPE wholemounts (3 hours in 4% paraformaldehyde [PFA], 30 minutes < 1 × phosphate-buffered saline [PBS] < 3 hours).

RESULTS

The new method consistently yields high-quality, intact retina and RPE wholemounts, with excellent structural integrity suitable for downstream imaging and molecular analyses. The technique significantly reduces preparation time. Optimal fixation conditions were identified, with 3 hours of fixation in 4% PFA and PBS incubation times between 30 minutes and 3 hours yielding the best results. The approach showed higher tissue integrity (80% vs. 45%) and improved staining quality of photoreceptor and ganglion cells. Additionally, the method is highly reproducible and effective for wholemount preparations from both young and older mice (6 and 12 months).

CONCLUSIONS

This study presents a significant advancement in the preparation of retina and RPE wholemounts. The simplicity, speed, and preservation of tissue integrity of the new method make it a valuable tool for ophthalmic disease research. Its potential applications include drug screening, gene therapy, and disease modeling, offering significant advantages in time efficiency, reproducibility, and the quality of morphological analysis.