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Nanyang Technological University

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform (RpEGEN) for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

2.5K Views

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13:12 min

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March 2nd, 2022

DOI :

10.3791/63658-v

March 2nd, 2022

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Lyndsay L. Leach¹, G. Burch Fisher², Jeffrey M. Gross¹^,³

¹Department of Ophthalmology, Louis J. Fox Center for Vision Restoration, University of Pittsburgh School of Medicine, ²Earth Research Institute, University of California, Santa Barbara, ³Department of Developmental Biology, University of Pittsburgh School of Medicine

Summary

This protocol describes the methodology to genetically ablate the retinal pigment epithelium (RPE) using a transgenic zebrafish model. Adapting the protocol to incorporate signaling pathway modulation using pharmacological compounds is extensively detailed. A MATLAB platform for quantifying RPE regeneration based on pigmentation was developed and is presented and discussed.

Explore More Videos

Retinal Pigment Epithelium

RPE Regeneration

RPE Degeneration

Pharmacological Treatment

Tissue Regeneration

Molecular Pathways

Ablation Protocol

Transgenic Larvae

Fluorescent Microscopy

Chapters in this video

0:05

Introduction

1:00

Screening Zebrafish Larvae for rpe65a:nfsB-eGFP and Genetic Ablation of the Retinal Pigment Epithelium

3:02

Incorporating Pharmacological Treatment into Zebrafish Retinal Pigment Epithelium Ablation Protocol

6:35

Confocal Microscope Z-stack Image Preprocessing in FIJI (ImageJ)

8:19

Quantification and Visualization of RPE Regeneration Using RpEGEN Scripts

10:42

Results: RPE Expression, Ablation, Regeneration Quantification

12:39

Conclusion

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