Name: Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes (Video) | JoVE Journal
Uploaded: 2014-05-03T14:00:00.000+00:00
Duration: 10 min 55 s
Description: Learn retrograde labeling of retinal ganglion cells in adult zebrafish using fluorescent dyes. This method enables quantitative analysis of RGC survival and degeneration following optic nerve injury.

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5 minutes

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.
<ol>
	<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 is 6 cm, width&#160;is 9 cm, height&#160;is 4 cm) in the middle and open a gap on it.</li>
	<li>Make a smooth globular surface on the end of the needle with light curing bond (Figure 1B) which will then be inserted into the fish mouth. Ensure that the surface of the needle is smooth to prevent the fish&#39;s mouth from being damaged.</li>
	<li>Add the half concentration of MS-222 (left chamber, length is 6 cm, width&#160;is 10 cm, height is 8 cm) and saline (right chamber, length is 4 cm, width&#160;is 10 cm, height is 8 cm) to the reservoirs as shown in Figure 1C. Note: Fresh MS-222 is recommended.</li>
	<li>Anesthetize fish in 0.03% MS-222 for 2 min.</li>
	<li>Place the fish in the gap of the sponge and fix the fish with two pairs of pins, inserted behind the pectoral fin and beside the cloacal pore, respectively11.</li>
	<li>Connect the fish&#8217;s mouth, MS-222, and rearing water with a T type tube as shown in Figure 1C.</li>
	<li>Switch on the tube connected to the MS-222.</li>
</ol>
2. Retrograde Label RGCs from the Right Tectum
NOTE: Prior to beginning, disinfect all apparatuses with 70-75% ethanol, ensuring that no ethanol remains.
<ol>
	<li>Rossing: slightly remove the skin over the whole skull with sclerectome (dorsal view of the skull is shown in Figures 2A and&#160;2B).</li>
	<li>Clearing: wash the skull with saline and then dry it with ear washing bulb.</li>
	<li>Initial corrosion: corrode the skull with the etchant for 10 sec.</li>
	<li>Clearing: completely wipe away the etchant with a cotton swab and then wash the area with saline and dry it.</li>
	<li>Protection: in order to protect the&#160;skull in&#160;other areas except the right tectum from excess corrosion, cover these areas with light curing bond and then cure it by exposure to blue light with portable illuminant.</li>
	<li>Complete corrosion: put the etchant in the central area of the right tectum again for complete etching for 2 min. Note: Since skulls of older fish are calcified, corrosion will take longer.</li>
	<li>Clearing: completely wipe away the etchant with a cotton swab and then wash the area with saline and dry it again.</li>
	<li>Skull opening: carefully remove the malacic skull (Figure 2C) with forceps to expose the right tectum (Figure 2D).</li>
	<li>Clearing: wipe the mucus and blood effusing from the hole with pieces of gelfoam and wash it with saline until the bleeding has stopped.</li>
	<li>Dye placement: put the dye (tissue-labeling paste) on the whole right tectum and cover it with gelfoam.</li>
	<li>Segregating: cover the hole with a sterile scale from the same animal.</li>
	<li>Sealing: place light curing bond onto the scale and then cure it with blue light again for 40 sec.</li>
	<li>Revival: wash the surface of the bond and revive the animal with saline.</li>
	<li>Nursing and breeding: Keep the fish in 28.5 &#176;C embryo medium (ZFIN) for 5 days and feed 2 times/day. Do not include fish whose dye fails to stay in the tectum for 5 days in the experiment. Note: Embryo medium (EM) solution is vital for fish survival and temperature is the key factor for DiI labeling.</li>
</ol>
3. Wholemounting Retina and Imaging
<ol>
	<li>Place the fish in a dark field for 2 hr&#160;before retina wholemount.</li>
	<li>Anesthetize the fish with ice water.</li>
	<li>Puncture a hole in the margin of cornea and make an eye cup by removing the cornea with venus scissors.</li>
	<li>Remove the lens and then flush the center of retina with ice-cold 1x&#160;PBS.</li>
	<li>Flush the gap between retina and sclera with ice-cold 1x&#160;PBS until no pigment is left.</li>
	<li>Cut the optic nerve at the disc.</li>
	<li>Keep the RGC layer upward and hold the retina with a self-made glass spoon (Figures 3A and&#160;3B). A dropper can also be used to transfer the retina.</li>
	<li>Put the retina in a drop of icy 30% glycerin on a slide with the RGC layer upward (Figure 3C).</li>
	<li>Remove the glycerin and then flatten the retina on the glass slide before cutting retina into four quadrants (Figure 3D). Keeping the retina unfolded with RGC layer upward will be helpful for flattening the retina.</li>
	<li>Drip icy 50% glycerin on the retina to wash away the fragments of pigment.</li>
	<li>Remove the glycerin and then drop 20 &#181;l of icy 75% glycerin on the retina.</li>
	<li>Cover the sample with a 1.8 cm x&#160;1.8 cm square coverslip.</li>
	<li>Seal the coverslip with nail polish. Immediate imaging of the retina is recommended.</li>
	<li>Non-interlace image the whole retina under a 20X&#160;object lens (NA=0.70) with DP72 CCD (each image is 1360 x&#160;1024 pixels). Each field has three different focuses.</li>
	<li>Extend the depth of each field with the tool of &#8220;extended depth of field&#8221; in the software.</li>
	<li>Assemble a virtual retina with the function of photomerge.</li>
	<li>Select three fields of 680 x&#160;512 pixels (actual&#160;area is 350 &#181;m by 264 &#181;m, 0.092 mm2) in each orientation of the retina to analyze average density of RGCs (Figure 4A).</li>
	<li>Obtain the area with the function of measurements &#8211; create a polygon feature in the image software.</li>
	<li>Calculate the total number of RGCs by the equation &#8220;RGCs number = destiny x&#160;area&#8221;&#160;9.</li>
</ol>

<ol><li>Wyatt, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+the+astray%2Frobo2+zebrafish+mutant+reveals+that+degenerating+tracts+do+not+provide+strong+guidance+cues+for+regenerating+optic+axons%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Staining+of+fluorogold-prelabeled+retinal+ganglion+cells+with+calcein-AM%3A+A+new+method+for+assessing+cell+vitality%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">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/pubmed?term=((Two+factors+secreted+by+the+goldfish+optic+nerve+induce+retinal+ganglion+cells+to+regenerate+axons+in+culture%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Survival+of+retinal+ganglion+cells+after+transection+of+the+optic+nerve+in+adult+cats%3A+a+quantitative+study+within+two+weeks%5BTitle%5D)+AND+%22Vis+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Retrograde+labeling+of+retinal+ganglion+cells+by+application+of+fluoro-gold+on+the+surface+of+superior+colliculus%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((Visual+system+scaling+in+teleost+fish%5BTitle%5D)+AND+%22J+Comp+Neurol%22%5BJournal%5D)">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/pubmed?term=((Changes+in+number+and+distribution+of+retinal+ganglion+cells+after+optic+nerve+crush+in+zebrafish%5BTitle%5D)+AND+%22Shi+Yan+Sheng+Wu+Xue+Bao%22%5BJournal%5D)">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/pubmed?term=((Ganglion+cell+regeneration+following+whole-retina+destruction+in+zebrafish%5BTitle%5D)+AND+%22Dev+Neurobiol%22%5BJournal%5D)">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/pubmed?term=((Neurogenesis+of+retinal+ganglion+cells+is+not+essential+to+visual+functional+recovery+after+optic+nerve+injury+in+adult+zebrafish%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?term=((Fluorescent+retrograde+neuronal+tracers+that+label+the+rat+facial+nucleus%3A+a+comparison+of+Fast+Blue%2C+Fluoro-ruby%2C+Fluoro-emerald%2C+Fluoro-Gold+and+DiI%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">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/pubmed?term=((Using+the+optokinetic+response+to+study+visual+function+of+zebrafish%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((Regulation+by+glycogen+synthase+kinase-3beta+of+the+arborization+field+and+maturation+of+retinotectal+projection+in+zebrafish%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">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/pubmed?term=((A+GFP-based+genetic+screen+reveals+mutations+that+disrupt+the+architecture+of+the+zebrafish+retinotectal+projection%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">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/pubmed?term=((Disruption+of+the+olfactoretinal+centrifugal+pathway+may+relate+to+the+visual+system+defect+in+night+blindness+b+mutant+zebrafish%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">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/pubmed?term=((Monitoring+of+Single-Cell+Responses+in+the+Optic+Tectum+of+Adult+Zebrafish+with+Dextran-Coupled+Calcium+Dyes+Delivered+via+Local+Electroporation%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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>

We have nothing to disclose.

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><tbody><tr><td>MS222</td><td>Sigma Aldrich, USA</td><td>E10521</td><td></td></tr><tr><td>DiI</td><td>Invitrogen, USA</td><td>N22880</td><td></td></tr><tr><td>Light curing bond</td><td>Heraeus Kulzer, Germany</td><td>Durafill bond</td><td></td></tr><tr><td>Gluma Etch</td><td>Heraeus Kulzer, Germany</td><td>Gluma Etch 35 Gel</td><td></td></tr><tr><td>Blue LED</td><td>Shenruo Medical Equipment Co., China</td><td>Power Blue Light Curing Unit</td><td></td></tr></tbody></table>

retrograde labeling of retinal ganglion cells in adult zebrafish with fluorescent dyes

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="Hyd...

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 ...

Adults.Debra Fish has a unique ability to regenerate neural system after injury such as hair cells, olfactory valve, T sland, spinal cord, and optic nerve. As the part of CNS optic nerve injury could induce RGC survival and degeneration. However, HG staining and antibody IHC could not specifically mark rrg disease with transgenic line marked all R GCs has not been constructed.In order to research the change of R GCs number after optic nerve injury in adult zebrafish, we successfully labeled all RGC by retrograde labeling RGC from the tum. Hi, I'm I'm, we are members of Laboratory Of Neuro Development and Repair School of Lexus University of Sales and Technology of China. And today we'll show you how to retrograde labeling RG CS in ABRA fish and get RG CS number in whole mountain retina.So Let's get it started. First, We need to build up A perfusion system to peruse anesthesia and the fresh water. You can find more details about the operators in the attached PDF document of this movie.Put the box above and make sure the fluid can flow smoothly from the box to the fish. Keep all operators disinfected during the Surgery. Prepare several speedballs and put them in the caves.Drip them with 75%ethanol and absolute ethanol. Prepare a neurore eye using a glass needle to deep it besides need geo Form acid etching, light curing bound tying forceps, jewelry forceps, acupuncture, ping blunt indeed forceps, scle rectum, scissors, coum swab. Salient blue LED glass needle with neurore eye on the end lighter and a 75%Ethanol.Anize the fish with 0.03%MS triple two. Then put it into the gap of the sponge. Add just the velocity of perfusion to about one drop per second.Put The needle into the fish mouth for oxygenation. Cover the fish with a piece of carpas to keep the skin wet. Now we turn to the top view for more details.First, remove the skin over the Whole sc. Wipe the surface with coum swab. Wash thecal with salient.Then dry it with ear washing bulb. Corrode the skull with the agent for 10 seconds, completely wipe away the agent. Then wash the area with salient and dry it Cover Areas.Accept the right tectum with light cure inbound for protection towards the incoming corrosion. Cure it with a period of 40 seconds exposure to blue light. Put agent on right Tum again for completely edge.For two minutes, completely wipe away the agent. Then wash the area with salient and dry it. Carefully open the skull with number five forceps.To expose the right tactile, absorb the cerebral spinal fluid infusing out from the wound with geo formm. Coat the dye over the whole right tectum. Then cover a piece of geo formm on D eye to attach it to the whole right tectum Tally.Cover the hole with the ster. Scale from the sand fish. Seal the wound with the light curing bound and Fix it with blue light.Switch off the anesthesia Solution. Revive the fish with fresh water. Usually we cut off their Things so they won't swing fast.In our lab, all surgeries are done by a skilled person. It takes 10 to 12 minutes per fish. About 20 to 30 fish can be labeled in half a day.Retina is not included in the Celia Zone. Make eye cup by removing the Celia zone and cornea with venous Scissors. Remove lens, then flush out aqueous fluid with I-C-P-B-S.Flush out pigment cut optic nerve at the disc. Remove The sclera to get a retina. Fixing the retina in 2%of PFA is optional.If imaging is not quickly following the separation, Clean the blood clouds and the pigment fragments before mounting. Transport The retina with a spoon made from curved cover glass. The ages are marked by lines.Put the retina In ic. 30%glycerin on a microscope.Slide. Keep RGCL Upward.Remove the glycerine, then cut the retina into four quadrants. Drop 20 microliter. I see 75%glycerin on the retina.Cover the sample with a square cover glass with a silence of 1.8 centimeter. Seal the cover glass with nail oil and the market. Observe the sample on fluorescence microscope via this Surgery.We successfully retrograde labeling RTCs in. Adapt zebra fish and get the RTCs number in whole mountain retina. By using light curing bound, we improve zebrafish survival rate that's making this method More effective.

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Research

JoVE Journal

Neuroscience

This video describes the method of retrograde labeling of RGC by applying fluoro-gold (FG) on the surface of superior colliculus (SC). Technique involves drilling the skull, aspirating the cortex, and applying gelatin sponge over entire dorsal surface of SC.

Retrograde Labeling of Retinal Ganglion Cells by Application of Fluoro-Gold on the Surface of Superior Colliculus

Key to understanding the morphogenetic processes that shape the early embryo is the ability to image cells at high resolution. We describe here a technique for labeling single cells or small clusters of cells in whole zebrafish embryos with membrane-targeted Green Fluorescent Protein.

Labeling and Imaging Cells in the Zebrafish Hindbrain

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Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The video demonstrates a method for culturing primary retinal ganglion cells from zebrafish embryos. Early-stage embryos undergo dissection to isolate the eyes, which are subsequently mechanically dissociated into a suspension of retinal ganglion cells. This cell suspension is cultured on coverslips coated with adhesive proteins to facilitate axon development.

Generation of Primary Retinal Ganglion Cell Cultures From Zebrafish Embryos

Source: Inal, M. A. et. al., Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes. J. Vis. Exp. (2020)This video demonstrates the method of retrograde labeling of Drosophila motor neurons using lipophilic dyes. The dye enters the axon, travels retrogradely, and labels the neurons green, allowing detailed visualization of their morphology and projections under a confocal microscope.

Retrograde Labeling of Drosophila Motor Neurons Using Fluorescent Lipophilic Dyes

Source: Laboissonniere, et. al. Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells. J. Vis. Exp. (2017). This video demonstrates a fluorescent tracing technique to identify retinal ganglion cell (RGC) subtypes in a transgenic mouse retina. The process involves securing the isolated retina in a recording chamber, maintaining cell viability with oxygenated perfusion, and targeting fluorescently labeled RGCs using a pipette with a tracer. By analyzing dendritic morphology and...

Identification of Mouse Retinal Ganglion Cell Subtypes Through Fluorescent Tracing

Source: Lahne, M., et.al. Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy. J. Vis. Exp. (2017)This video demonstrates live-cell imaging of Muller glial nuclear migration in agarose-embedded zebrafish retinal explants following light-induced photoreceptor damage. Tumor necrosis factor-alpha (TNF-α) released by damaged photoreceptors activates Muller glial cells, triggering nuclear migration through the inner and outer nuclear layers for...

Live Cell Imaging of Muller Glial Nuclear Migration During Zebrafish Retinal Regeneration

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

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

Learn retrograde labeling of retinal ganglion cells in adult zebrafish using fluorescent dyes. This method enables quantitative analysis of RGC survival and degeneration following optic nerve injury.

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

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