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>
	<li>Lieschke, G., Currie, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+human+disease:+zebrafish+swim+into+view.">Animal models of human disease: zebrafish swim into view.</a> Nat. Rev. Genet. 8 (5), 353-367 (2007).</li><li>Ma, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+Pde6+reduces+cell+body+Ca2++transients+within+photoreceptors.">Loss of Pde6 reduces cell body Ca2+ transients within photoreceptors.</a> Cell Death Dis. 4 (9), e797 (2013).</li><li>Niethammer, P., Grabher, C., Look, A., Mitchison, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tissue-scale+gradient+of+hydrogen+peroxide+mediates+rapid+wound+detection+in+zebrafish.">A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish.</a> Nature. 459 (7249), 996-999 (2009).</li><li>Andrews, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualising+apoptosis+in+live+zebrafish+using+fluorescence+lifetime+imaging+with+optical+projection+tomography+to+map+FRET+biosensor+activity+in+space+and+time.">Visualising apoptosis in live zebrafish using fluorescence lifetime imaging with optical projection tomography to map FRET biosensor activity in space and time.</a> J. Biophotonics. 9 (4), 414-424 (2016).</li><li>Kioka, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+intramitochondrial+ATP+levels+identifies+G0/G1+switch+gene+2+as+a+positive+regulator+of+oxidative+phosphorylation.">Evaluation of intramitochondrial ATP levels identifies G0/G1 switch gene 2 as a positive regulator of oxidative phosphorylation.</a> Proc. Natl. Acad. Sci. U.S.A. 111 (1), 273-278 (2014).</li><li>Muto, A., Ohkura, M., Abe, G., Nakai, J., Kawakami, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-Time+Visualizationof+Neuronal+Activity+during+Perception.">Real-Time Visualizationof Neuronal Activity during Perception.</a> Curr. Biol. 23 (4), 307-311 (2013).</li><li>Stewart, A., Braubach, O., Spitsbergen, J., Gerlai, R., Kalueff, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+models+for+translational+neuroscience+research:+from+tank+to+bedside.">Zebrafish models for translational neuroscience research: from tank to bedside.</a> Trends Neurosci. 37 (5), 264-278 (2014).</li><li>Zou, S. -. Q., Yin, W., Zhang, M. -. J., Hu, C. -. R., Huang, Y. -. B., Hu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+the+optokinetic+response+to+study+visual+function+of+zebrafish.">Using the optokinetic response to study visual function of zebrafish.</a> J. Vis. Exp. (36), e1742 (2010).</li><li>Neuhauss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+genetic+approaches+to+visual+system+development+and+function+in+zebrafish.">Behavioral genetic approaches to visual system development and function in zebrafish.</a> J. Neurobiol. 54 (1), 148-160 (2002).</li><li>Stearns, G., Evangelista, M., Fadool, J., Brockerhoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Mutation+in+the+Cone-Specific+pde6+Gene+Causes+Rapid+Cone+Photoreceptor+Degeneration+in+Zebrafish.">A Mutation in the Cone-Specific pde6 Gene Causes Rapid Cone Photoreceptor Degeneration in Zebrafish.</a> J. 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M., Holzhausen, L., Kramer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+an+optogenetic+pH+sensor+reveals+that+protons+mediate+lateral+inhibition+in+the+retina.">Imaging an optogenetic pH sensor reveals that protons mediate lateral inhibition in the retina.</a> Nat. Neurosci. 17 (2), 262-268 (2014).</li><li>Van Hook, M., Thoreson, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+Whole-cell+Recordings+from+Photoreceptors+and+Second-order+Neurons+in+an+Amphibian+Retinal+Slice+Preparation.">Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation.</a> J. Vis. Exp. (76), e50007 (2013).</li><li>Connaughton, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+retinal+slice+preparation.">Zebrafish retinal slice preparation.</a> Meth. Cell Sci. 25 (1/2), 49-58 (2003).</li><li>Kennedy, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+zebrafish+cone+photoreceptor-specific+promoter+and+genetic+rescue+of+achromatopsia+in+the+nof+mutant.">Identification of a zebrafish cone photoreceptor-specific promoter and genetic rescue of achromatopsia in the nof mutant.</a> Invest. Ophthalmol. Vis. Sci. 48 (2), 522-529 (2007).</li><li>George, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptojanin+1+is+required+for+endolysosomal+trafficking+of+synaptic+proteins+in+cone+photoreceptor+inner+segments.">Synaptojanin 1 is required for endolysosomal trafficking of synaptic proteins in cone photoreceptor inner segments.</a> PLoS ONE. 9 (1), e84394 (2014).</li><li>Shin, J., Chen, J., Solnica-Krezel, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+homologous+recombination-mediated+genome+engineering+in+zebrafish+using+TALE+nucleases.">Efficient homologous recombination-mediated genome engineering in zebrafish using TALE nucleases.</a> Development. 141 (19), 3807-3818 (2014).</li><li>Giarmarco, M., Lindsay, K., Du, J., Cleghorn, W., Brockerhoff, S., Hurley, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Real-time+Glucose+Uptake+Reveals+Distinct+Transport+Among+Cell+Types.">Imaging Real-time Glucose Uptake Reveals Distinct Transport Among Cell Types.</a> Invest. Ophthalmol. Vis. Sci. , (2015).</li><li>Kanow, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+adaptations+of+the+retina+and+retinal+pigment+epithelium+support+a+metabolic+ecosystem+in+the+vertebrate+eye.">Biochemical adaptations of the retina and retinal pigment epithelium support a metabolic ecosystem in the vertebrate eye.</a> eLife. 6, (2017).</li><li>Giarmarco, M., Cleghorn, W., Sloat, S., Hurley, J., Brockerhoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+Maintain+Distinct+Ca2++Pools+in+Cone+Photoreceptors.">Mitochondria Maintain Distinct Ca2+ Pools in Cone Photoreceptors.</a> J. Neurosci. 37 (8), 2061-2072 (2017).</li><li>Uckermann, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+staining+by+vital+dyes+of+M&#252;ller+glial+cells+in+retinal+wholemounts.">Selective staining by vital dyes of M&#252;ller glial cells in retinal wholemounts.</a> Glia. 45 (1), 59-66 (2003).</li><li>Johnson, S., Rabinovitch, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+imaging+of+excised+tissue+using+vital+dyes+and+confocal+microscopy.">Ex vivo imaging of excised tissue using vital dyes and confocal microscopy.</a> Current protocols in cytometry. , (2012).</li><li>Kulkarni, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Ca2++Dynamics+in+Cone+Photoreceptor+Axon+Terminals+of+the+Mouse+Retina.">Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina.</a> J. Vis. Exp. (99), (2015).</li></ol>

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">
	<tbody>
		<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 ...

The overall goal of this procedure is to prepare fresh slices of Zebrafish retinas for experiments that require live imaging of fluorescent proteins or biosensors. This method can help answer key questions in the field of vision research, such as the physiological and metabolic roles of calcium ions in specific cell types and cellular compartments within a retina. The main advantage of this technique is that it provides spatial and temporal information about key metabolites and signaling molecules in live tissues.To make it possible to remove the retinal pigment epithelium, or RPE, from the Zebrafish, dark adapt the fish for one hour prior to making tissue slices. For a slicing chamber, paint lines of nail polish onto a slide. Using clear nail polish, paint narrow lines to create a rectangle.Repaint the polish once when dry. If the chamber will be used for static imaging, or injection experiments with minimal solution flow, then use a syringe to apply two parallel strips of petroleum jelly to a coverslip, one centimeter long and 0.5 centimeters apart. Now prepare the blades.First, clean a double-edged razor with ethanol and allow it to air-dry. Then use scissors to cut it into approximately one centimeter pieces. Next, place the slicing chamber on the stage of the tissue slicer, center it horizontally on the stage, and mark the long edge with permanent marker for alignment.Then, load a piece of blade onto the slicer arm. Secure the blade, so the edge is over the middle of the slide and runs parallel with the slide surface, but does not touch the nail polish. Then lower the blade arm using a one quarter turn.Now, try slicing filter paper. Remount the blade if needed. To proceed, using the syringe deposit a single, small dot of jelly in a 10 centimeter dish, about 1.5 centimeters from the midpoint.Then press the dry side of a coverslip into the jelly using forceps. Next, put another small dot of jelly about one centimeter from the inlet edge of the imaging chamber. Now, use a 20 gauge needle to make two one centimeter long thin parallel strips of petroleum jelly lengthwise along the center of the slicing chamber about one centimeter apart.Then transfer the Zebrafish to a dish and cervically dislocate the fish without decapitation. Now, use a wire loop to loosen the connective tissue around one eye and then pull the eye forward gently. Next, carefully cut the white optic nerve under the eye using microscissors, but do not cut the back of the eye.To proceed, transfer the eye to a dish of cold Ringer's solution on ice. Then harvest the other eye. Continue working under red light until all of the RPE is removed.Next, transfer one eye into a drop of cold Ringer's solution on a glass slide on a petri dish. Then place the dish under a low-powered dissection microscope to dissect the eye cup under red light. First, pierce the cornea with fine forceps.Next, use forceps and scissors to gently remove pieces of clear brittle cornea and silvery sclera. Then remove and discard the lens, and most of the sclera to isolate the eye cup. With minimal handling remove any pieces of fat and sclera that remain attached to the eye cup without removing the retina.If the retina separates from the RPE, then proceed with extra caution to avoid damaging the delicate retina. Now, reposition the eye cup with open side down and cut it into thirds or quarters with a new blade. Discard the pieces of eye cup that are too curved to flatten out.These pieces are often too small to be useful in any case. To mount the retina sections, wet a piece of filter paper with Ringer's solution and use flat forceps to place it next to the eye cup pieces. Then immerse the filter paper and tissues in cold Ringer's solution.Next, use forceps to position retina pieces on top of filter paper with the RPE and photoreceptors up. Gently handle the edges of the eye cup pieces with fine forceps to orient them into a single line at the center of the filter paper. Now flatten the retina by using forceps to lift the filter paper with retina attached and setting it on a dry paper towel.For about three seconds, let the force of liquid wicking into the paper towel flatten the retina. Process all of the retina pieces in this manner, just don't let them dry out. Now, apply a drop of Ringer's solution and use fine forceps to peel away any remaining black sheets of RPE from the tissue.Peel gently, and if the retina lifts off the paper, wick away more solution as before and it should flatten again. Now transfer the paper with retina pieces to a slide and trim the paper into a rectangle leaving about 0.5 centimeters on either side of the row of tissue. Then, move the filter paper to the prepared slicing chamber.Push the long filter paper edges into the thin petroleum jelly lines using forceps, and then immerse the retinas in three to four drops of cold Ringer's solution. First, transfer the preparation to the tissue slicer stage. Position the long edge along the marked line and secure the chamber ends to the stage with laboratory tape.Then, starting at one end, cut the retina and filter paper using firm, gentle pressure on the slicing arm. Check that the first slice was cut fully, and then proceed to use the micrometer to cut slices that are approximately 400 microns thick. Next, transfer the chamber with sliced sections to the petri dish containing the prepared coverslip, which will serve as a imaging ladder.Flood the dish with cold Ringer's solution and place it on the stage of the dissecting microscope. Now, under low magnification, use the forceps to secure a strip and move it onto the imaging ladder by sliding the underlying petri dish. Rotate the slices 90 degrees and bury the filter paper edges into the jelly.It is important to keep the retina submerged and to avoid making any direct contact with the retina. Next, reposition the slices in the ladders so that the retina layers are clearly visible. For the slices at each end of the ladder, make the retina face inward toward the other slices to minimize motion during injection or flow.Discard any slices that do not adhere well to the paper. Now, stain the tissue. Meanwhile, prepare the imaging chamber and injection apparatus.First, flush the tubing with Ringer's solution, until no air bubbles remain. Then, attach the tubing to the imaging chamber inlet and close the stop cock. Then, refill the syringe with the solution for injection and reattach it to the tubing.Now, use forceps to transfer the prepared imaging ladder to the imaging chamber. Press it into the dot of jelly near the inlet edge and fill the chamber with Ringer's solution to cover the slices. Then proceed with imaging.Using the described techniques, perfectly transversed slices can be imaged through all of the retinal layers. When slightly rotated, bundles of the outer segments will be visible. Using PI staining, dead cells can be eliminated from analysis.Healthy PI-negative photoreceptors have a stereotypical polarized elongated morphology. When in oxygenated Ringer's, they can be imaged for up to four hours. One imaging strategy is to visualize the dynamics of cone photoreceptor endoplasmic reticulum relative to the nucleus.Using tissues that express GFP, and the cone ER, while counterstaining with a nuclear dye. A similar strategy can be employed to view the actin cytoskeleton with tissue expressing GFP-fused actin. Using time-lapsed imaging, cellular dynamics such as cytosolic calcium fluctuations in a cone photoreceptor can be observed.For example, while isotonically depleting sodium in the imaging chamber, GCaMP can be used to visualize rising cytosolic calcium concentrations. By quantifying the fluorescence responses from single cone outer segments, cell bodies and synapses, the intracellular calcium dynamics can be determined during pharmacological manipulations and so forth. While attempting this procedure, it's important to remember to use red light before the RPE is removed.Carefully flatten eye cups before slicing, and keep the slices submerged at all times. Once mastered, tissue dissection, slicing, and preparation for imaging can be done in less than 30 minutes. After watching this video, you should have a good understanding of how to prepare retinal slices from Zebrafish to study metabolism, and physiology in specific cell types in cellular compartments.

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Research

JoVE Journal

Neuroscience

9.0K visualizações.  University of Washington. The overall goal of this procedure is to prepare fresh slices of Zebrafish retinas for experiments that require live imaging of fluorescent proteins or biosensors. This method can help answer key questions in the field of vision research, such as the physiological and metabolic roles of calcium ions in specific cell types and cellular compartments within a retina. The main advantage of this technique is that it provides spatial and temporal information about key metabolites and signaling mole...