Preparing Fresh Retinal Slices from Adult Zebrafish for <em>Ex Vivo</em> Imaging Experiments

Michelle M. Giarmarco; Whitney M. Cleghorn; James B. Hurley; Susan E. Brockerhoff

doi:10.3791/56977

Preparazione fette fresche della retina dallo Zebrafish adulto per Ex Vivo Imaging esper...

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JoVE Journal Neuroscience

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

Preparazione fette fresche della retina dallo Zebrafish adulto per Ex Vivo Imaging esperimenti

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11:01 min

May 9, 2018

DOI: 10.3791/56977-v

Michelle M. Giarmarco¹, Whitney M. Cleghorn¹, James B. Hurley^1,2, Susan E. Brockerhoff^1,2

¹Department of Biochemistry,University of Washington, ²Department of Ophthalmology,University of Washington

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Overview

This article presents a protocol for preparing fresh slices of zebrafish retina to facilitate live imaging using fluorescent biosensors. The methodology aims to enhance understanding of calcium ion roles in specific retinal cell types and compartments, providing crucial spatial and temporal information on biological processes.

Key Study Components

Area of Science

Neuroscience
Retinal biology
Imaging techniques

Background

Imaging retinal tissue allows for single-cell analysis not achievable via biochemistry.
Zebrafish are a valuable model for understanding vertebrate retinal functions.
The technique provides insights into calcium ion dynamics in retinal cells.

Purpose of Study

To explore the physiological and metabolic roles of calcium ions in zebrafish retinal cells.
To develop a reliable method for obtaining live retinal slices for imaging.
To enhance understanding of cellular processes in vision research.

Methods Used

Ex vivo imaging platform utilizing fresh slices of zebrafish retina.
The biological model includes retinal cells and their cellular compartments.
Critical steps include dark adaptation of fish, careful dissection, and immersion in cold Ringer’s solution.
The method outlines the preparation of slicing chambers, slicing techniques, and positioning of retina slices for imaging.

Main Results

The method enabled the observation of calcium ion dynamics in live retinal tissues.
Key insights into metabolic signaling and visual processing in distinct retinal cell types were gathered.
Mechanistic understanding of calcium ion effects on retinal functionality was enhanced.

Conclusions

This study establishes a protocol that facilitates live imaging in zebrafish retina, advancing the understanding of vision-related cellular processes.
The insights gained can contribute to broader implications for studying neuronal mechanisms in retinal health and disease.

Frequently Asked Questions

What are the advantages of using zebrafish for retinal studies?

Zebrafish offer a transparent model, allowing direct observation of live retinal processes and cellular behaviors, making them ideal for imaging studies.

How is the retinal pigment epithelium (RPE) removed in the protocol?

The fish is dark adapted prior to dissection, and careful techniques are employed to ensure the RPE is removed without damaging the delicate retina.

What types of imaging data are obtained with this method?

The method allows for real-time visualization of calcium dynamics and other biological processes in various retinal cell types using fluorescent biosensors.

Can this technique be adapted for use in other types of tissues?

While specific modifications would be necessary, the basic principles of live tissue slicing and imaging can be adapted for other biological tissues.

What are key limitations of the slicing method?

Careful handling and precise techniques are crucial to avoid damage to the retina; excessive handling may lead to complications in imaging quality.

What are the implications of the findings for vision research?

Understanding calcium dynamics in retinal cells can provide insights into vision-related disorders and help develop therapeutic strategies.

Formazione immagine del tessuto retinico può fornire informazioni di cella singola che non possono essere raccolti dai metodi biochimici tradizionali. Questo protocollo descrive la preparazione delle fette della retina dallo zebrafish per imaging confocale. Fluorescenti geneticamente codificati sensori o indicatore coloranti permettono la visualizzazione di numerosi processi biologici in tipi distinti delle cellule retiniche.

L'obiettivo generale di questa procedura è quello di preparare fette fresche di retina di pesce zebra per esperimenti che richiedono l'imaging dal vivo di proteine fluorescenti o biosensori. Questo metodo può aiutare a rispondere a domande chiave nel campo della ricerca sulla vista, come i ruoli fisiologici e metabolici degli ioni calcio in specifici tipi di cellule e compartimenti cellulari all'interno di una retina. Il vantaggio principale di questa tecnica è che fornisce informazioni spaziali e temporali sui metaboliti chiave e sulle molecole di segnalazione nei tessuti vivi.

Per rendere possibile la rimozione dell'epitelio pigmentato retinico, o RPE, dal pesce zebra, adattare il pesce scuro per un'ora prima di fare fette di tessuto. Per una camera di affettatura, dipingere linee di smalto per unghie su un vetrino. Usando lo smalto trasparente, dipingi linee strette per creare un rettangolo.

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