Agradecemos a drs. Richard H. Masland e Akimichi Kaneko por nos dar conselhos e sugestões valiosas para estabelecer o procedimento da preparação fatia horizontal da retina.

<ol><li> Prepare um bloco de agar (30 mg / ml, ou seja 3%, em água destilada). Dissolver e microondas-lo primeiro e coloque em uma travessa de vidro. Mantenha o prato no frigorífico a 4 º C. Dica: O bloco de ágar deve ser preparado com antecedência. </li><li> Prepare baixa temperatura de fusão-agarose gel (25 mg / ml, ou seja, 2,5%, em média). Dissolver e microondas. Mantê-lo no banho de água quente a 35 o C. Dica: Mantenha o gel agarose não muito quente. Se for muito quente, ele leva muito tempo a ser solidificado, e que o gel pode matar a retina . </li><li> Eutanásia do animal enuclear, um globo ocular e abri-lo como uma ocular. Para liquefazer humor vítreo, a ocular aberto é embebido por 10 min em hialuronidase no meio (0,07 mg / ml), se necessariamente. </li><li> Corte o agar em um bloco (por exemplo, 1 cm x 1,5 cm para retina mouse) e firmemente fixados em um palco vibratome por cola instantânea. Manter o bloco de ágar molhado. </li><li> Coloque a lâmina de 5 graus na slicer. Gentilmente cortar a superfície superior do bloco de agar na velocidade de aprox. 50 mm / seg, freq. 50 Hz. Dica: Certifique-se que a superfície superior do bloco de agar é totalmente lisa e plana. Vários cortes (pelo menos 3 vezes) pode ser necessária para obter a superfície lisa e plana do bloco de ágar. </li><li> Lavar a ocular com o meio sem hialuronidase. Isolar a retina de um epitélio pigmentar e coloque-o fotorreceptor cabeça para baixo em um pedaço de papel de filtro. Para firmemente anexar a retina ao papel de filtro, vácuo da retina no papel de filtro várias vezes. Dica: Certifique-se que a retina está ligado ao filtro de papel completamente plana. Depois de colocar a retina no filtro de papel, corte o excesso de papel filtro em torno da retina (Fig.2A). O excesso de papel de filtro, muitas vezes impede uma lâmina de ir para a camada apropriada da retina. </li><li> Limpe cuidadosamente o excesso de médios no bloco de agar e coloque cuidadosamente a retina sobre o papel de filtro sobre o bloco de ágar. Para firmemente colocar o papel de filtro sobre o bloco de ágar, anexar cola instantânea na borda do papel de filtro. Dica: Para evitar rachadura unexpectable, coloque a retina como frente possível na superfície superior do bloco de ágar. </li><li> Delicadamente despeje a baixa temperatura de fusão do gel agarose sobre a retina. Espere por 1 min e derrame delicadamente o meio sobre ele para resfriá-lo. O gel de agarose torna-se solidificou rapidamente. Dica: Muito quente agarose gel mata a retina. </li><li> Levante a posição da lâmina de várias centenas M acima da superfície superior do bloco de agar e gentilmente cortar o topo da solidificou agarose gel. </li><li> Mais abaixo da posição da lâmina. Cortar a retina ao nível do proximal de um terço da camada nuclear interna (aprox. entre 200 e 250 M acima da superfície superior do bloco de agar). Dica: Excesso de velocidade da lâmina irá danificar a retina severamente. Aprox. 50 mm / s (50 Hz) é apropriada. </li><li> Observe que as células amácrinas estão agora viradas para baixo. Com cuidado, pegue a fatia da retina juntamente com o gel de agarose solidificou e colocá-lo virado para cima em uma câmara ou um prato. Dica: Como a retina do rato é pequeno e ondulado, é difícil obter uma fatia perfeita horizontal em uma camada apropriada. Uma fatia &quot;oblíquas&quot; pode ser um método alternativo para obter a fatia relativa plano da retina. Para obter a fatia oblíqua, colocar outro papel de filtro entre a retina sobre o papel de filtro eo bloco de ágar, conforme mostrado na Fig.2B. Embora você não pode esperar que uma fatia horizontal perfeita, há sempre algumas áreas onde soma de células amácrinas está na superfície da fatia. </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>

Todos os protocolos experimentais foram conduzidos de acordo com os Institutos Nacionais de Saúde diretrizes para uso animal.

Existem várias vantagens importantes da preparação fatia horizontal da retina. Primeiro, a morfologia das células que se expandem horizontalmente dendrites, como as células amácrinas e células horizontal, é preservada. Tem sido demonstrado que há mais de 20 tipos de células amácrinas na retina (MacNeil e Masland, 1998), de modo que é muito importante para comparar a morfologia das células com a propriedade fisiológica. Em segundo lugar, em toda a retina de montagem, a extensa cobertura do endfeets células glia...

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		<div>
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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<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>
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horizontal slice preparation of the retina

Tradicionalmente a fatia vertical e toda a preparação para montagem da retina têm sido usados ​​para estudar a função dos circuitos da retina. No entanto, muitos dos neurônios da retina, tais como as células amácrinas, expandir as suas dendrites horizontalmente, de modo que a morfologia das células é suposto ser severamente danificado nas fatias verticais. Em toda a preparação de montagem, especialmente para patch-clamp gravações, os neurônios da retina na camada do meio não são facilmente acessíveis devido à extensa cobertura de células gliais endfeets (célula Mueller) s. Aqui, descrevemos a novela corte método para preservar a morfologia dendrítica de neurônios da retina intacta. A fatia foi feito horizontalmente na camada interna da retina usando um cortador de vibratome após a retina foi incorporado na baixa temperatura de fusão agarose gel. Nessa preparação fatia horizontal da retina, estudamos a função dos neurônios da retina comparado com sua morfologia, usando patch-clamp gravação, técnica de imageamento de cálcio, imunocitoquímica, e de uma única célula RT-PCR.

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

Horizontal Slice Preparation of the Retina

Tradicionalmente a fatia vertical e toda a preparação para montagem da retina têm sido usados ​​para estudar a função dos circuitos da retina. Aqui, descrevemos a novela corte método para preservar a morfologia dendrítica de neurônios da retina intacta.

Preparação Slice horizontal da 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 visualizações.  Dalhousie University. So the legendary is here, and this is a picture paper.

Vídeo: 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.

Claro / escuro Teste de transição para Ratos

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, ...

Biologia

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.

Cultura organotípicas de Retina Coelho Adulto

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.

Seccionamento fina de Preparações Slice para Imunohistoquímica

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

Organotypic Slice Culture of E18 Rat Brains

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice. 

Cultura Slice organotípicas de E18 cérebros de ratos

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

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.

Preparação de Gene Gun Bullets e Transfection biobalística de neurônios em cultura Slice

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.

De célula inteira Recordings of Light Evocados Excitatórios Correntes Synaptic na Slice Retinal

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.

Evisceração de Vítreo Mouse e Retina de Análises Proteômica

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 ...

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.

Em Eletroporação vivo de Morpholinos na Retina Zebrafish Adulto

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.

Em Eletroporação Ovo na retina de pinto embrionário

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.