Name: preparing fresh retinal slices from adult zebrafish for ex vivo imaging experiments
Uploaded: 2018-05-09T12:30:00
Description: Watch this Scientific Journal Video about Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments at JoVE.com

We thank Ralph Nelson and Daniel Possin for thoughtful guidance while developing this protocol, and Eva Ma, Ashley George and Gail Stanton for generation of stable transgenic zebrafish lines. The work was supported by NSF GRFP 2013158531 to M.G., NIH NEI 5T32EY007031 to W.C. and M.G., and EY026020 to J.H. and S.B.

All animal experiments were approved by the University of Washington Institutional Animal Care and Use Committee.
1. Preparing Animals and Equipment
NOTE: The retinal pigment epithelium (RPE) is a dark sheet of tissue surrounding the outside of the retina whose pigmentation can obscure retinal features and damage the tissue when confocal imaging ex vivo. In darkness, the RPE of zebrafish is retracted away from the retina; dark adapt fish to facilitate future ...

<ol>
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Ex vivo imaging of fresh zebrafish retinal slices has proven to be a versatile tool for studying photoreceptor biology20,21,22, and is unique in that it enables analysis of single cells in a mature, fully differentiated retina. With practice, it is possible to conduct multiple experiments with tissue from a single fish, even using serial slices from the same part of the retina. In addition to the challenges and suggesti...

The zebrafish (Danio rerio) has become widely used in medical and basic scientific research1, owing to its small size, rapid development and vertebrate organ systems. The natural transparency of zebrafish larvae combined with established methods for transgenesis have enabled detailed visualization of cellular processes in a living animal. A number of genetically encoded fluorescent biosensors have been targeted to specific zebrafish cells to detect Ca2+ 2, hydrogen peroxide3, apoptotic activation4 and ATP5.
In vivo imaging of zebrafish larvae has led to breakthroughs in the field of neuroscience, including mapping of brain circuitry6 and drug development for central nervous system disorders7. Zebrafish are well suited for vision research because their retinas feature the laminar structure and neuron types of higher vertebrates, and they display robust visual behaviors8,9. Several types of retinal degenerations analogous to human disease have been modeled successfully and studied in zebrafish10,11, including live imaging of individual photoreceptors degenerating within a retina2,12.
While in vivo larval zebrafish imaging is a valuable tool, it becomes more challenging as fish grow and develop pigmentation, and some pharmacological treatments cannot permeate an entire animal. Further, certain cellular processes change with development and age, making later time points critical for understanding function and the progression of disease in adult animals. Biochemical methods such as immunoblot, quantitiative PCR, O2 consumption, and metabolomic analyses can provide important clues about biology of the retina as a whole, but it is difficult to discern contributions of individual cell types affected by disease. Imaging isolated retinal tissue ex vivo bypasses these issues, and while imaging flat mounted retinas affords a view of the outer retina13, deeper inner retinal features are obscured. Transverse retinal slices, such as those presented in fixed immunohistochemical analyses, enable a clear view of all layers and cell types but only offer a single snapshot of the dynamic processes involved in normal function and disease.
Here, we present a method for generating ex vivo transverse retinal slices from adult zebrafish for imaging. It is similar to methods for preparing amphibian and zebrafish retinal slices for electrophysiological and morphological studies14,15, with important modifications for time lapse imaging ex vivo using confocal microscopy. Fluorescence responses of biosensors or dyes in slices are monitored in real time with a confocal microscope while delivering pharmacological agents using perfusion. While the method was developed for imaging photoreceptors, it may be feasible to use it for visualizing M&#252;ller cells, bipolar cells, horizontal cells, amacrine cells, or retinal ganglion cells with appropriate fluorescent markers. Additionally, slices can be loaded with fluorescent cell-permeable dyes to report cell viability, vesicular transport, mitochondrial function, or redox state. This versatile preparation allows visualization of a wide range of subcellular processes throughout the retina, including Ca2+ dynamics, signal transduction and metabolic state.

<table border="0" cellpadding="0" cellspacing="0" width="1153">
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		<tr height="40">
			<td height="40" style="height:40px;width:332px;">zebrafish</td>
			<td style="width:239px;">Univeristy of Washington South Lake Union Aquatics Facility</td>
			<td style="width:181px;"></td>
			<td style="width:401px;">stocks maintained in-house as stable transgenic lines</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">petroleum jelly</td>
			<td style="width:239px;">Fisher Scientific</td>
			<td style="width:181px;">19-090-843</td>
			<td style="width:401px;">for petroleum jelly syringe</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">3-mL slip tip syringe</td>
			<td style="width:239px;">Fisher Scientific</td>
			<td style="width:181px;">14-823-436</td>
			<td style="width:401px;">for petroleum jelly syringe</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">20g 3.8 cm slip tip needle</td>
			<td style="width:239px;">Fisher Scientific</td>
			<td style="width:181px;">14-826-5B</td>
			<td style="width:401px;">for petroleum jelly syringe</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">plain 7 cm X 2.5 cm microscope slide</td>
			<td style="width:239px;">Fisher Scientific</td>
			<td style="width:181px;">12-550-A3</td>
			<td style="width:401px;">for eyecup dissection, slicing chamber</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">Seche Vite clear nail polish</td>
			<td style="width:239px;">Amazon</td>
			<td style="width:181px;">B00150LT40</td>
			<td style="width:401px;">for slicing chamber</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">18 mm X 18 mm #1 glass coverslips</td>
			<td style="width:239px;">Fisher Scientific</td>
			<td>12-542A</td>
			<td style="width:401px;">for imaging ladders</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">unflavored dental wax</td>
			<td style="width:239px;">Amazon</td>
			<td style="width:181px;">B01K8WNL5A</td>
			<td style="width:401px;">for imaging ladders</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">double edge razor blades</td>
			<td style="width:239px;">Stoelting</td>
			<td style="width:181px;">51427</td>
			<td style="width:401px;">for tissue slicing</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">tissue slicer with digital micrometer</td>
			<td style="width:239px;">Stoelting</td>
			<td style="width:181px;">51415</td>
			<td style="width:401px;">for tissue slicing</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:332px;">filter paper - white gridded mixed cellulose, 13 mm diameter, 0.45 &#181;m pore size</td>
			<td style="width:239px;">EMD Millipore</td>
			<td style="width:181px;">HAWG01300</td>
			<td style="width:401px;">filter paper for mounting retinas</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">10 cm petri dish</td>
			<td style="width:239px;">Fisher Scientific</td>
			<td style="width:181px;">FB0875712</td>
			<td style="width:401px;">for fish euthanasia, dissection, imaging ladder assembly</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">15 cm plain-tipped wood applicator stick</td>
			<td style="width:239px;">Fisher Scientific</td>
			<td style="width:181px;">23-400-112</td>
			<td style="width:401px;">for wire eye loop tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">30g (0.25 mm diameter) tungsten wire</td>
			<td style="width:239px;">Fisher Scientific</td>
			<td style="width:181px;">AA10408G6</td>
			<td style="width:401px;">for wire eye loop tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">D-glucose</td>
			<td style="width:239px;">Sigma Aldrich</td>
			<td style="width:181px;">G8270</td>
			<td style="width:401px;">component of supplement stock solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">sodium L-lactate</td>
			<td style="width:239px;">Sigma Aldrich</td>
			<td style="width:181px;">L7022</td>
			<td style="width:401px;">component of supplement stock solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">sodium pyruvate</td>
			<td style="width:239px;">Sigma Aldrich</td>
			<td style="width:181px;">P2256</td>
			<td style="width:401px;">component of supplement stock solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-glutamine</td>
			<td style="width:239px;">Sigma Aldrich</td>
			<td style="width:181px;">G3126</td>
			<td style="width:401px;">component of supplement stock solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;L-glutathione, reduced</td>
			<td style="width:239px;">Sigma Aldrich</td>
			<td style="width:181px;">G4251</td>
			<td style="width:401px;">component of supplement stock solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-ascorbic acid</td>
			<td style="width:239px;">Sigma Aldrich</td>
			<td style="width:181px;">A5960</td>
			<td style="width:401px;">component of supplement stock solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td style="width:239px;">Sigma Aldrich</td>
			<td style="width:181px;">S7653</td>
			<td style="width:401px;">component of Ringer&#39;s solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCl</td>
			<td style="width:239px;">Sigma Aldrich</td>
			<td style="width:181px;">P9333</td>
			<td style="width:401px;">component of Ringer&#39;s solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CaCl2 &#183; 2H2O</td>
			<td style="width:239px;">Sigma Aldrich</td>
			<td style="width:181px;">C3881</td>
			<td style="width:401px;">component of Ringer&#39;s solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaH2PO4</td>
			<td style="width:239px;">Sigma Aldrich</td>
			<td style="width:181px;">S8282</td>
			<td style="width:401px;">component of Ringer&#39;s solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MgCl2 &#183; 6H2O</td>
			<td style="width:239px;">Sigma Aldrich</td>
			<td style="width:181px;">M0250</td>
			<td style="width:401px;">component of Ringer&#39;s solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td style="width:239px;">Sigma Aldrich</td>
			<td style="width:181px;">H3375</td>
			<td style="width:401px;">component of Ringer&#39;s solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris base</td>
			<td style="width:239px;">Fisher Scientific</td>
			<td style="width:181px;">BP152</td>
			<td style="width:401px;">component of Na+-free Ringer&#39;s solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6 N HCl</td>
			<td style="width:239px;">Fisher Scientific</td>
			<td style="width:181px;">02-003-063</td>
			<td style="width:401px;">component of Na+-free Ringer&#39;s solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KH2PO4</td>
			<td style="width:239px;">Sigma Aldrich</td>
			<td style="width:181px;">P5655</td>
			<td style="width:401px;">component of Na+-free Ringer&#39;s solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">50 mL conical centrifuge tube</td>
			<td style="width:239px;">Denville Scientific</td>
			<td style="width:181px;">C1062-P</td>
			<td style="width:401px;">container for Ringer&#39;s solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">Vannas scissors - 8 cm, angled 5 mm blades</td>
			<td style="width:239px;">World Precision Instruments</td>
			<td style="width:181px;">501790</td>
			<td style="width:401px;">micro-scissors for eyecup dissection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:332px;">Swiss tweezers - #5, 11 cm, straight, 0.06 X 0.07 mm tips</td>
			<td style="width:239px;">World Precision Instruments</td>
			<td style="width:181px;">504510</td>
			<td style="width:401px;">fine forceps for eyecup dissection and slice manipulation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">single edge razor blades</td>
			<td style="width:239px;">Fisher Scientific</td>
			<td style="width:181px;">12-640</td>
			<td style="width:401px;">for eyecup dissection and trimming filter paper</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">EMD Millipore filter forceps</td>
			<td style="width:239px;">Fisher Scientific</td>
			<td style="width:181px;">XX6200006P</td>
			<td style="width:401px;">flat forceps for handling wet filter paper</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:332px;">C12 558/568 BODIPY</td>
			<td style="width:239px;">Fisher Scientific</td>
			<td style="width:181px;">D3835</td>
			<td style="width:401px;">stains live cell nuclei; incubate 5 &#181;g/mL for 15 min at room temperature</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:332px;">propidium iodide (PI)</td>
			<td style="width:239px;">Fisher Scientific</td>
			<td style="width:181px;">P3566</td>
			<td style="width:401px;">stains dead cell nuclei; incubate 5 &#181;g/mL for 20 min at room temperature</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:332px;">Hoechst 33342</td>
			<td style="width:239px;">Fisher Scientific</td>
			<td style="width:181px;">62249</td>
			<td style="width:401px;">stains live cell nuclei; incubate 5 &#181;g/mL for 20 min at room temperature</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:332px;">Tetramethylrhodamine, methyl ester (TMRM)</td>
			<td style="width:239px;">Fisher Scientific</td>
			<td style="width:181px;">T668</td>
			<td style="width:401px;">stains functional, negatively-charged mitochondria; incubate 1 nM for 30 min at room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">tissue perfusion chamber</td>
			<td style="width:239px;">Cell MicroControls</td>
			<td style="width:181px;">BT-1-18/BT-1-18BV [-SY]</td>
			<td style="width:401px;">imaging chamber for injection or perfusion</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:332px;">2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (NBDG)</td>
			<td style="width:239px;">Fisher Scientific</td>
			<td style="width:181px;">N13195</td>
			<td style="width:401px;">fluorescent glucose analog adminitered orally to zebrafish 30 min prior to euthanasia</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:332px;">Olympus laser scanning confocal microscope</td>
			<td style="width:239px;">Olympus</td>
			<td style="width:181px;">FV1000</td>
			<td style="width:401px;">confocal microscope for visualizing fluorescence of slices at single-cell resolution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:332px;">Carbonyl cyanide 3-chlorophenylhydrazone (CCCP)</td>
			<td style="width:239px;">Sigma Aldrich</td>
			<td style="width:181px;">C2759</td>
			<td style="width:401px;">experimental reagent which ablates mitochondrial respiration; treat slices to a final concentration of 1 &#181;M</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">miniature aspirator positioner</td>
			<td style="width:239px;">Cell MicroControls</td>
			<td style="width:181px;">FL-1</td>
			<td style="width:401px;">for perfusion</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:332px;">perfusion manifold, gas bubbler manifold, flow valve, 60cc syringe holder</td>
			<td style="width:239px;">Warner Instruments</td>
			<td style="width:181px;">various</td>
			<td style="width:401px;">for perfusion</td>
		</tr>
	</tbody>
</table>

preparing fresh retinal slices from adult zebrafish for ex vivo imaging experiments

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding diseases, and future therapies hinge on fundamental understandings about how different retinal cells function normally. Gaining such information with biochemical methods has proven difficult because contributions of particular cell types are diminished in the retinal cell milieu. Live retinal imaging can provide a view of numerous biological processes on a subcellular level, thanks to a growing number of genetically encoded fluorescent biosensors. However, this technique has thus far been limited to tadpoles and zebrafish larvae, the outermost retinal layers of isolated retinas, or lower resolution imaging of retinas in live animals. Here we present a method for generating live ex vivo retinal slices from adult zebrafish for live imaging via confocal microscopy. This preparation yields transverse slices with all retinal layers and most cell types visible for performing confocal imaging experiments using perfusion. Transgenic zebrafish expressing fluorescent proteins or biosensors in specific retinal cell types or organelles are used to extract single-cell information from an intact retina. Additionally, retinal slices can be loaded with fluorescent indicator dyes, adding to the method&#39;s versatility. This protocol was developed for imaging Ca2+ within zebrafish cone photoreceptors, but with proper markers it could be adapted to measure Ca2+ or metabolites in M&#252;ller cells, bipolar and horizontal cells, microglia, amacrine cells, or retinal ganglion cells. The retinal pigment epithelium is removed from slices so this method is not suitable for studying that cell type. With practice, it is possible to generate serial slices from one animal for multiple experiments. This adaptable technique provides a powerful tool for answering many questions about retinal cell biology, Ca2+, and energy homeostasis.

Stable positioning and transverse orientation of slices are key to successful imaging with injection or perfusion of pharmacological agents. Carefully examine and reposition slices prior to confocal imaging as needed to ensure all retinal layers are visible (Figure 2A, slice ii). If a slice is rotated slightly forward (Figure 2A, slice iii), bundles of outer segments will be visible and small adjustments can be made with forceps ...

Watch this Scientific Journal Video about Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments at JoVE.com

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

Imaging retinal tissue can provide single-cell information that cannot be gathered from traditional biochemical methods. This protocol describes preparation of retinal slices from zebrafish for confocal imaging. Fluorescent genetically encoded sensors or indicator dyes allow visualization of numerous biological processes in distinct retinal cell types.

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

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding ...

Research

JoVE Journal

Neuroscience

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

We present a method developed specifically to image the whole Drosophila brain during ongoing behavior such as walking. Head fixation and dissection are ...

Isolation of Adult Human Astrocyte Populations from Fresh-Frozen Cortex Using Fluorescence-Activated Nuclei Sorting

The complexity of human astrocytes remains poorly defined in primary human tissue, requiring better tools for their isolation and molecular ...

Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and ...

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. ...

Mouse Retina OCT Fibergram Alignment with Confocal Images

Alignment of Visible-Light Optical Coherence Tomography Fibergrams with Confocal Images of the Same Mouse Retina

Advancements in In Vivo and Ex Vivo Retinal Imaging for Improved Glaucoma Diagnosis and Treatment (Video) | JoVE

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has ...

Monitoring Epileptiform Events in Drosophila via Calcium Imaging

Ex Vivo Calcium Imaging for Drosophila Model of Epilepsy

Insights into the Techniques and Findings of Recent Advancements in Epilepsy Research (Video) | JoVE

Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals ...