This is supported by 973 MOST grant (Grant No. 2011CB504402, 2012CB947602), National Natural Science Foundation of China (Grant No. 91132724, U1332136) and the 'Hundred Talents Project' of Chinese Academy of Science. The protocol was approved by the Committee on the Ethics of Animal Experiments of the USTC (Permit Number: USTCACUC1103013).

1. Construct the Surgery Apparatus
NOTE: To ensure the fish remains alive during and after the operation, drip anesthesia solution ethyl 3-aminobenzoate methanesulfonate (MS-222, or Tricaine) at a half concentration (0.015%) with a velocity of 1 drop/sec&#160;through the fish&#39;s mouth using a homebuilt drip system shown in Figure 1.
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	<li>Make a box as shown in Figure 1A (length is 28 cm, width&#160;is 10 cm, height&#160;is 5 cm), put a sponge (length...

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	<li>Wyatt, C., 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=Analysis+of+the+astray/robo2+zebrafish+mutant+reveals+that+degenerating+tracts+do+not+provide+strong+guidance+cues+for+regenerating+optic+axons.">Analysis of the astray/robo2 zebrafish mutant reveals that degenerating tracts do not provide strong guidance cues for regenerating optic axons.</a> J Neurosci. 30, 13838-13849 (2010).</li><li>Grieshaber, P., Lagreze, W. A., Noack, C., Boehringer, D., Biermann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staining+of+fluorogold-prelabeled+retinal+ganglion+cells+with+calcein-AM:+A+new+method+for+assessing+cell+vitality.">Staining of fluorogold-prelabeled retinal ganglion cells with calcein-AM: A new method for assessing cell vitality.</a> J Neurosci Methods. 192, 233-239 (2010).</li><li>Schwalb, J. 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=Two+factors+secreted+by+the+goldfish+optic+nerve+induce+retinal+ganglion+cells+to+regenerate+axons+in+culture.">Two factors secreted by the goldfish optic nerve induce retinal ganglion cells to regenerate axons in culture.</a> J Neurosci. 15, 5514-5525 (1995).</li><li>Watanabe, M., Inukai, N., Fukuda, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+of+retinal+ganglion+cells+after+transection+of+the+optic+nerve+in+adult+cats:+a+quantitative+study+within+two+weeks.">Survival of retinal ganglion cells after transection of the optic nerve in adult cats: a quantitative study within two weeks.</a> Vis Neurosci. 18, 137-145 (2001).</li><li>Chiu, K., Lau, W. M., Yeung, S. C., Chang, R. C., So, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrograde+labeling+of+retinal+ganglion+cells+by+application+of+fluoro-gold+on+the+surface+of+superior+colliculus.">Retrograde labeling of retinal ganglion cells by application of fluoro-gold on the surface of superior colliculus.</a> J Vis Exp. , (2008).</li><li>Bakken, T. E., Stevens, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+system+scaling+in+teleost+fish.">Visual system scaling in teleost fish.</a> J Comp Neurol. 520, 142-153 (2012).</li><li>Zhou, L. X., Wang, Z. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+number+and+distribution+of+retinal+ganglion+cells+after+optic+nerve+crush+in+zebrafish.">Changes in number and distribution of retinal ganglion cells after optic nerve crush in zebrafish.</a> Shi Yan Sheng Wu Xue Bao. 35, 159-162 (2002).</li><li>Sherpa, T., 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=Ganglion+cell+regeneration+following+whole-retina+destruction+in+zebrafish.">Ganglion cell regeneration following whole-retina destruction in zebrafish.</a> Dev Neurobiol. 68, 166-181 (2008).</li><li>Zou, S., Tian, C., Ge, S., 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=Neurogenesis+of+retinal+ganglion+cells+is+not+essential+to+visual+functional+recovery+after+optic+nerve+injury+in+adult+zebrafish.">Neurogenesis of retinal ganglion cells is not essential to visual functional recovery after optic nerve injury in adult zebrafish.</a> PLoS One. 8, (2013).</li><li>Choi, D., Li, D., Raisman, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+retrograde+neuronal+tracers+that+label+the+rat+facial+nucleus:+a+comparison+of+Fast+Blue,+Fluoro-ruby,+Fluoro-emerald,+Fluoro-Gold+and+DiI.">Fluorescent retrograde neuronal tracers that label the rat facial nucleus: a comparison of Fast Blue, Fluoro-ruby, Fluoro-emerald, Fluoro-Gold and DiI.</a> J Neurosci Methods. 117, 167-172 (2002).</li><li>Zou, S. Q., 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=Using+the+optokinetic+response+to+study+visual+function+of+zebrafish.">Using the optokinetic response to study visual function of zebrafish.</a> J Vis Exp. , (2010).</li><li>Tokuoka, H., Yoshida, T., Matsuda, N., Mishina, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+by+glycogen+synthase+kinase-3beta+of+the+arborization+field+and+maturation+of+retinotectal+projection+in+zebrafish.">Regulation by glycogen synthase kinase-3beta of the arborization field and maturation of retinotectal projection in zebrafish.</a> J Neurosci. 22, 10324-10332 (2002).</li><li>Xiao, T., Roeser, T., Staub, W., Baier, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+GFP-based+genetic+screen+reveals+mutations+that+disrupt+the+architecture+of+the+zebrafish+retinotectal+projection.">A GFP-based genetic screen reveals mutations that disrupt the architecture of the zebrafish retinotectal projection.</a> Development. 132, 2955-2967 (2005).</li><li>Li, L., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+the+olfactoretinal+centrifugal+pathway+may+relate+to+the+visual+system+defect+in+night+blindness+b+mutant+zebrafish.">Disruption of the olfactoretinal centrifugal pathway may relate to the visual system defect in night blindness b mutant zebrafish.</a> Journal of Neuroscience. 20, 1883-1892 (2000).</li><li>Kassing, V., Engelmann, J., Kurtz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+Single-Cell+Responses+in+the+Optic+Tectum+of+Adult+Zebrafish+with+Dextran-Coupled+Calcium+Dyes+Delivered+via+Local+Electroporation.">Monitoring of Single-Cell Responses in the Optic Tectum of Adult Zebrafish with Dextran-Coupled Calcium Dyes Delivered via Local Electroporation.</a> PLoS One. 8, (2013).</li></ol>

Retrograde labeling of RGCs is important to research RGCs survival in mammalian animals, but it has not been used in zebrafish. The alternative methods, HE staining7 and antibody staining8, are not gold standards for counting RGCs number, and transgenic&#160;lines with all RGCs labeled has not yet been constructed12,13. In this video, we introduce a method to retrograde label RGCs in the retina of adult zebrafish. Though about 1% of axons project to olfactory bulb or...

As adult zebrafish have a strong ability to regenerate axons after optic nerve injury1, a proper method to count the whole RGCs is essential to evaluate RGCs survival and regeneration2. Based on the methods of retrograde labeling RGCs in mammalian and goldfish3-5, we constructed the method to label RGCs from the tectum in adult zebrafish. For adult zebrafish, two critical technical concerns should be noted: the skull of adult zebrafish is very small6; they live in a water environment. Here, we treat the skull with etchant which minimizes the hazards associated with drilling5. Then, we seal the hole with light curing bond which improves animal survival after surgery.
Previously, several other techniques were adopted to count RGCs number in indirect ways. HE staining in the retina sections labels all types of cells in the RGCL7. Antibody labeling in the whole retina, such as islet-1, can also label amacrine cells8. Though retrograde label from the optic nerve stump can label all RGCs in the retina, it cannot be adopted in the crush model because it causes extra injury to the optic nerve. Taking advantage of retrograde label from the tectum, we have researched RGCs survival and regeneration in optic nerve crush. Results show that almost all RGCs survived and over 90% of RGCs regenerated to the tectum at the first week in the crush model9.
In order to successfully label all RGCs, DiI paste was chosen after comparison with several other commercial dyes10. Firstly, it is especially designed for in vivo tissue labeling. Secondly, it is a lipophilic dye which cannot diffuse in water. Additionally, this fluorescence can persist for a long time which makes it an excellent candidate for RGCs survival research.

<table border="0" cellpadding="0" cellspacing="0" width="608">
	<tbody>
		<tr height="37">
			<td height="37" style="height:37px;width:152px;">MS222</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:152px;">E10521</td>
			<td style="width:152px;">USA</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:152px;">DiI</td>
			<td style="width:152px;">Invitrogen</td>
			<td style="width:152px;">N22880</td>
			<td style="width:152px;">USA</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:152px;">lightcuring bond</td>
			<td style="width:152px;">Heraeus Kulzer&#160;</td>
			<td style="width:152px;">Durafill bond</td>
			<td style="width:152px;">Germany</td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;width:152px;">Gluma Etch</td>
			<td style="width:152px;">Heraeus Kulzer&#160;</td>
			<td style="width:152px;">Gluma Etch 35 Gel</td>
			<td style="width:152px;">Germany</td>
		</tr>
		<tr height="73">
			<td height="73" style="height:73px;width:152px;">Blue LED</td>
			<td style="width:152px;">Shenruo Medical Equipment Co.</td>
			<td style="width:152px;">Power Blue Light Curing Unit</td>
			<td style="width:152px;">China</td>
		</tr>
	</tbody>
</table>

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 survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

As Figures 4B-D show, the number of DiI+ cells is two thirds of DAPI+ cells in RGCL. In a normal retina, a montage image of a whole retina (Figure 4E) shows that DiI+ RGCs are distributed over the entire retina but in a regenerated retina (Figure 4F), as RGCs in the central area have not regenerated to their target at the first week, they could not be labeled.
<img alt="Fig...

Watch this Scientific Journal Video about Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes at JoVE.com

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

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

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 ...

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9.4K Views.  University of Science and Technology of China. We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.