Wir danken Drs. Richard H. Masland und Akimichi Kaneko, dass sie uns wertvolle Hinweise und Anregungen an das Verfahren der horizontalen Scheibe Vorbereitung der Netzhaut zu etablieren.

<ol><li> Bereiten Sie eine Agar-Block (30 mg / ml, dh 3%, in destilliertem Wasser). Auflösen und Mikrowelle es zuerst und gießen Sie sie in einer Glasschale. Halten Sie das Gericht in einem Kühlschrank bei 4 ° C. Tipp: Die Agar-Block sollte im Vorfeld vorbereitet werden. </li><li> Bereiten Sie bei niedrigen Temperaturen schmelzende Agarosegel (25 mg / ml, dh 2,5%, in Medium). Auflösen und Mikrowelle es. Halten Sie es in dem heißen Wasserbad bei 35 o C. Tipp: Halten Sie das Agarosegel nicht zu heiß. Wenn es zu heiß ist, dauert es eine lange Zeit verfestigt werden, und das Gel kann die Netzhaut zu töten . </li><li> Euthanize das Tier, entkernen einen Augapfel, und öffnen Sie es als eine Augenmuschel. Zu verflüssigen Glaskörper, ist die geöffnete Augenmuschel für 10 min in Hyaluronidase in das Medium (0,07 mg / ml) getränkt, wenn nötig. </li><li> Schneiden Sie das Agar in einen Block (zB, 1 cm x 1,5 cm für Maus Netzhaut) und fest auf einem Vibratom Bühne von Sekundenkleber befestigt. Halten Sie die Agarblock nass. </li><li> Legen Sie das Blatt bei 5 Grad an der Schneidemaschine. Vorsichtig schnitt die Oberfläche des Agar-Block mit einer Geschwindigkeit von ca.. 50 mu m / sec, Freq. 50 Hz. Tipp: Stellen Sie sicher, dass die Oberfläche des Agar-Block völlig glatt und eben ist. Mehrere Schnitte (mindestens 3-mal) erforderlich sein, um die glatte und ebene Oberfläche des Agar-Block zu bekommen. </li><li> Spülen Sie die Augenmuschel mit dem Medium ohne Hyaluronidase. Isolieren Sie die Netzhaut aus einer Pigmentepithel und legen Sie sie Photorezeptor-Seite nach unten auf ein Stück Filterpapier. Um fest an der Netzhaut auf das Filterpapier, Vakuum der Netzhaut auf dem Filterpapier mehrmals. Tipp: Stellen Sie sicher, dass die Netzhaut auf das Filterpapier vollständig flach ist beigefügt. Nachdem Sie eine Netzhaut setzen auf dem Filterpapier, schneiden Sie die überschüssige Filterpapier Umgebung der Netzhaut (Abb. 2a). Der Überschuss Filterpapier verhindert oft eine Klinge aus, die in der entsprechenden Schicht der Netzhaut. </li><li> Wischen Sie das überschüssige Medium auf die Agar-Block und vorsichtig die Netzhaut auf dem Filterpapier auf die Agar-Block. Um einen festen Platz das Filterpapier auf die Agar-Block legen Sekundenkleber am Rand des Filterpapiers. Tipp: Um zu vermeiden, Unerwartete crack, Ort der Netzhaut wie vor als möglich auf der Oberseite des Agar-Block. </li><li> Vorsichtig gießen Sie die niedrigen Temperaturen schmelzende Agarosegel über die Netzhaut. Warten Sie 1 min und gießen Sie leicht das Medium auf, um es abzukühlen. Das Agarosegel verfestigt schnell. Tipp: Zu heiß Agarosegel tötet die Netzhaut. </li><li> Nehmen Sie die Klinge der Lage, mehrere hundert um über die Oberfläche des Agar-Block und sanft schneiden Sie die Spitze des erstarrten Agarosegel. </li><li> Weiter unten die Klinge Position. Cut der Netzhaut auf der Ebene des proximalen ein Drittel der inneren Körnerschicht (ca. zwischen 200 und 250 pm oberhalb der oberen Oberfläche des Agar-Block). Tipp: Zu viel Geschwindigkeit des Blattes wird die Netzhaut stark beschädigen. Ca. 50 mu m / sec (50 Hz) geeignet ist. </li><li> Beachten Sie, dass die Amakrinzellen jetzt verdeckt sind. Sorgfältig nehmen die Scheibe der Netzhaut zusammen mit der erstarrten Agarosegel und legen Sie sie offen in eine Kammer oder ein Gericht. Tipp: Da die Maus Netzhaut ist winzig und wellig, ist es schwierig, eine perfekte horizontale Scheibe an einer geeigneten Schicht zu erhalten. Eine &quot;schräge&quot; Scheibe kann eine alternative Methode, um die relative flache Scheibe der Netzhaut zu erhalten. Um die schrägen Scheibe, anders Filterpapier in zwischen der Netzhaut auf dem Filterpapier und die Agar-Block wie in Fig. 2B gezeigt. Obwohl Sie nicht erwarten können eine perfekte horizontale Scheibe, gibt es immer einige Bereiche, in denen amakrinen Zelle soma ist auf der Oberfläche der Scheibe. </li></ol>

<ol>
	<li>Azuma, T., Enoki, R., Iwamuro, K., Kaneko, A., Koizumi, A., Koizumi, A., TC, J. a. k. o. b. s., Masland, R. H., MA, M. a. c. N. e. i. l., RH, M. a. s. l. a. n. d. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+spatiotemporal+patterns+of+dendritic+Ca2++signals+in+goldfish+retinal+amacrine+cells.">Multiple spatiotemporal patterns of dendritic Ca2+ signals in goldfish retinal amacrine cells.</a> Brain Res. , 64-73 (2004).</li></ol>

Alle experimentellen Protokolle wurden nach Angaben des National Institutes of Health Richtlinien für Tierversuche durchgeführt.

Es gibt mehrere wichtige Vorteile der horizontalen Scheibe Vorbereitung der Netzhaut. Zunächst wird die Morphologie der Zellen, die Dendriten erweitern horizontal wie Amakrinzellen und horizontale Zellen, erhalten. Es hat sich gezeigt, dass es über 20 Arten von Amakrinzellen in der Netzhaut (MacNeil und Masland, 1998), so dass es ganz wichtig ist, die Morphologie der Zellen mit der physiologische Eigenschaft zu vergleichen. Zweitens, in der ganzen-mount Netzhaut verhindert, dass die umfangreiche Berichterstattung von Gliaz...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Agar</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A-1296</td>
<td>&nbsp;</td>
<tr>
<td>Agarose L</td>
<td>&nbsp;</td>
<td>Wako</td>
<td>317-01182</td>
<td>Low-temperature melting agarose </td>
</tr>
<tr>
<td>Hyaluronidase</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>Type I-S, 300 units/mg</td>
</tr>
<tr>
<td>Medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>in mM: 102 NaCl, 3.1 KCl, 2.0 CaCl2, 1.0 MgCl2, 23 NaHCO3, and 10 glucose, maintained at pH 7.8, or, Ames&#x2019; Solution (Sigma)</td>
</tr>
<tr>
<td>Microwave</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Hot water bath</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>35 C</td>
</tr>
<tr>
<td>Vibratome slicer</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>VT1000S</td>
<td>&nbsp;</td>
<tr>
<td>MF-Membrane filters</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>HAWP01300</td>
<td>Filter paper , pore size 0.45 um</td>
</tr>
<tr>
<td>Dissection tools</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>forceps, blade, pinch, twizzer, etc.</td>
</tr>
<tr>
<td>Instant glue</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>eg., Alonalpha, Krazy Glu</td>
</tr>
<tr>
<td>50 ml syringe</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>syringe filter</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>SX0001300</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

horizontal slice preparation of the retina

Traditionell wird der vertikale Scheiben schneiden und die ganze-mount Vorbereitung der Netzhaut verwendet worden, um die Funktion der Netzhaut Schaltkreise zu untersuchen. Allerdings sind viele der retinalen Nervenzellen, wie Amakrinzellen, erweitern ihre Dendriten horizontal, so dass die Morphologie der Zellen soll zu stark in den vertikalen Scheiben beschädigt werden. In der ganzen-mount Vorbereitung, insbesondere für Patch-Clamp-Aufnahmen sind Netzhautneuronen in der mittleren Schicht nicht leicht zugänglich durch die umfangreiche Berichterstattung von Gliazellen (Mueller-Zelle) s endfeets. Hier beschreiben wir den Roman Slicing-Verfahren, um die dendritischen Morphologie der retinalen Nervenzellen intakt zu erhalten. Die Scheibe wurde horizontal an der inneren Schicht der Netzhaut mit einem Vibratom Allesschneider gemacht, nachdem der Netzhaut in der Tieftemperatur-Agarose-Gel eingebettet war. In diesem horizontalen Scheibe Vorbereitung der Netzhaut, untersuchten wir die Funktion der Netzhaut Neuronen im Vergleich zu ihrer Morphologie, mit Patch-Clamp-Aufzeichnung, Kalzium-Imaging-Technik, Immunzytochemie und Einzelzell-RT-PCR.

Watch this Scientific Journal Video about Horizontal Slice Preparation of the Retina at JoVE.com

Horizontal Slice Preparation of the Retina

Traditionell wird der vertikale Scheiben schneiden und die ganze-mount Vorbereitung der Netzhaut verwendet worden, um die Funktion der Netzhaut Schaltkreise zu untersuchen. Hier beschreiben wir den Roman Slicing-Verfahren, um die dendritischen Morphologie der retinalen Nervenzellen intakt zu erhalten.

Horizontale Scheiben Vorbereitung der Retina

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of ...

So the legendary is here, and this is a picture paper.

horizontal-slice-preparation-of-the-retina

Research

JoVE Journal

Biology

15.7K Aufrufe.  Dalhousie University. So the legendary is here, and this is a picture paper.

Serie: Horizontal Slice Preparation of the Retina

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

Hell / Dunkel Transition Test für Mäuse

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

Forschung

Biologie

Organotypic Culture of Adult Rabbit Retina

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with imaging techniques, genetic manipulation, and electrophysiological recording. Here we present a simple interphase tissue culture system for adult rabbit retina that requires no specialized equipment and very little maintenance. We demonstrate the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells. Rabbit retinas cultured this way can be kept alive for up to 6 days with very little changes of the overall anatomical structure or the morphology of individual ganglion- and amacrine cells.

This article demonstrates the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells.

Organotypischen Kultur der Adult Kaninchen Retina

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with ...

Thin Sectioning of Slice Preparations for Immunohistochemistry

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been preserved, freshly removed, or excised but kept viable, as in slice preparations of brain tissue.  Subsequent microscopic studies of this material can be challenging, as the tissue samples may be difficult to handle.  Demonstrated here is a method for obtaining thin cryostat sections of tissue with a thickness that may range from 0.2-5.0 mm.  We routinely cut 400 micron thick Vibratome brain slices serially into 5-10 micron coronal cryostat sections.  The slices are typically first used for electrophysiology experiments and then require microscopic analysis of the cytoarchitecture of the region from which the recordings were observed.  We have constructed a simple device that allows controlled and reproducible preparation and positioning of the tissue slice.  This device consists of a cylinder 5 cm in length with a diameter of 1.2 cm, which serves as a freezing stage for the slice.  A ring snugly slides over the cylinder providing walls around the slice allowing the tissue to be immersed in freezing compound (e.g., OCT).  This is then quickly frozen with crushed dry ice and the resulting  wafer  can be position easily for cryostat sectioning.  Thin sections can be thaw-mounted onto coated slides to allow further studies to be performed, such as various staining methods, in situ hybridization, or immunohistochemistry, as demonstrated here.

The present method allows reproducible cryostat sectioning of small, difficult-to-manage, tissue pieces, such as biopsies and brain slices. We utilize a simple aluminum freezing stage to facilitate handling of tissue and a standard cryostat to routinely produce 5-10 micron serial sections from 400 micron thick brain slices.

Dünnschnitt der Scheibe Vorbereitungen für die Immunhistochemie

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been ...

Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed protocol for biolistic transfection of rat hippocampal slices, from the initial preparation of DNA coated bullets to the final shooting of the organotypic slice cultures using a gene gun. Gene gun transfection is an efficient and easy means of transfecting neurons and is especially useful for fluorescently labeling a small subset of cells in tissue slice. In this video, we first outline the steps required to coat gold particles with DNA. We next demonstrate how to line the inside of plastic tubing with the gold/DNA bullets, and how to cut this tubing to obtain the plastic cartridges for loading into the gene gun. Finally, we perform biolistic transfection of rat hippocampal slice cultures, demonstrating handling of the Bio-Rad Helios gene gun, and offering trouble shooting advice to obtain healthy and optimally transfected tissue slices.  

We describe a method for preparing DNA coated gold bullets and demonstrate the use of such bullets to biolistically transfect neurons in cultured hippocampal slices.

Vorbereitung der Gene Gun und Aufzählungszeichen Biolistic Transfektion von Neuronen in Scheiben Kultur

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed ...

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we will guide you through the process of performing whole-cell recordings of light evoked currents of individual cells in the retinal slice preparation. We use the aquatic tiger salamander as an animal model. We begin by describing the dissection of the eye and show how slices are mounted for electrophysiological recordings. Once the slice is placed in the recording chamber, we demonstrate how to perform whole-cell voltage clamp recordings. We then project visual stimuli onto the photoreceptors in the slice to elicit light-evoked current responses. During the recording we perfuse the slice with pharmacological agents, whereby an 8-channel perfusion system allows us to quickly switch between different agents.  The retinal slice preparation is widely used for patch clamp recordings in the retina, in particular to study amacrine or bipolar cells, which are not accessible in a whole-mount preparation.

This video shows the process of whole-cell voltage clamp recordings in the retinal slice of the aquatic tiger salamander. We demonstrate the preparation of the slice as well as how to perform patch clamp recordings during visual stimulation of the retina.

Ganzzellableitungen of Light evozierten exzitatorischen synaptischen Ströme in der Netzhaut Scheiben

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we ...

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is a highly aqueous extracellular matrix overlying the retina where intraocular as well as extraocular proteins accumulate during disease.1-3 Abnormal interactions between vitreous and retina underlie several diseases such as retinal detachment, proliferative diabetic retinopathy, uveitis, and proliferative vitreoretinopathy.1,4 The relative mouse vitreous volume is significantly smaller than the human vitreous (Figure 1), since the mouse lens occupies nearly 75% of its eye.5 This has made biochemical studies of mouse vitreous challenging. In this video article, we present a technique to dissect and isolate the mouse vitreous from the retina, which will allow use of transgenic mouse models to more clearly define the role of this extracellular matrix in the development of vitreoretinal diseases.

The dissection technique illustrates evisceration of the vitreous, retina, and lens from the mouse eye, separation by centrifugation, and characterization with protein assays.

Ausweiden der Maus Glaskörper und Netzhaut für Proteomik-Analysen

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is ...

The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic by-products. This balance is achieved by the movement of blood through the microcirculation, which encompasses the smallest branches of the vascular supply throughout all tissues and organs. Arterioles branch from arteries to form networks that control the distribution and magnitude of oxygenated blood flowing into the multitude of capillaries intimately associated with parenchymal cells. Capillaries provide a large surface area for diffusional exchange between tissue cells and the blood supply. Venules collect capillary effluent and converge as they return deoxygenated blood towards the heart. To observe these processes in real time requires an experimental approach for visualizing and manipulating the living microcirculation.
The cremaster muscle of rats was first used as a model for studying inflammation using histology and electron microscopy post mortem1,2. 
The first in vivo report of the exposed intact rat cremaster muscle investigated microvascular responses to vasoactive drugs using reflected light3. However 
curvature of the muscle and lack of focused illumination limited the usefulness of this preparation. The major breakthrough entailed opening the muscle, 
detaching it from the testicle and spreading it radially as a flat sheet for transillumination under a compound microscope4. While shown to be a valuable 
preparation to study the physiology of the microcirculation in rats5 and hamsters6, the cremaster muscle in mice7 has proven particularly useful in dissecting 
cellular pathways involved in regulating microvascular function8-11 and real-time imaging of intercellular signaling12. 
The cremaster muscle is derived from the internal oblique and transverse abdominus muscles as the testes descend through the inguinal canal13. 
It serves to support (Greek: cremaster = suspender) and maintain temperature of the testes. As described here, the cremaster muscle is prepared as a thin flat sheet 
for outstanding optical resolution. With the mouse maintained at a stable body temperature and plane of anesthesia, surgical preparation involves freeing the muscle 
from surrounding tissue and the testes, spreading it onto transparent pedestal of silastic rubber and securing the edges with insect pins while irrigating it 
continuously with physiological salt solution. The present protocol utilizes transgenic mice expressing GCaMP2 in arteriolar endothelial cells. GCaMP2 is a 
genetically encoded fluorescent calcium indicator molecule12. Widefield imaging and an intensified charge-coupled device camera enable in vivo study of 
calcium signaling in the arteriolar endothelium.

A tissue preparation is described for visualization and experimental manipulation of the living microcirculation. In anesthetized male mice, the thin, highly vascularized cremaster muscle is prepared for intravital microscopy to study microvascular networks including arterioles, capillaries and venules. This preparation is readily adapted for rats and hamsters.

Die Maus Cremaster Muscle Vorbereitung Intravital Imaging der Mikrozirkulation

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

In-vivo-Elektroporation von Morpholinos in die Adult Zebrafisch Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In Ovo Electroporation in Embryonic Chick Retina

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is comparatively very easy to manipulate the chick embryonic retina using recombinant DNA/RNA technology. Electroporation of DNA/RNA constructs into the embryonic retina have a great advantage to study gene regulation in retinal stem/progenitor cells during retinal development. Different type of assays such as reporter gene assay, gene over-expression, gene knock down (shRNA) etc. can be performed using the electroporation technique. This video demonstrates targeted retinal injection and in ovo electroporation into the embryonic chick retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). Here we show a rapid and convenient in ovo electroporation technique whereby a plasmid DNA that expresses green fluorescent protein (GFP) as a marker is directly delivered into the chick embryonic subretinal space and followed by electric pulses to facilitate DNA uptake by retinal stem/progenitor cells. The new method of retinal injection and electroporation at E4 allows the visualization of all retinal cell types, including the late-born neurons1, which has been difficult with the conventional method of injection and electroporation at E1.52.

In Ovo Electroporation in Embryonic Chick Retina

The overall goal of this video is to show how to perform targeted retinal injection and in ovo electroporation of DNA/RNA constructs into the chick embryonic retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). This technique is very useful to study gene expression, gene regulation, and morphological change in developing chick retina.

In ovo Elektroporation in embryonalen Hühnchenretina

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.