우리는 DRS 감사합니다. 망막의 수평 슬라이스 준비 절차를 확립하기 위해 우리에게 귀중한 조언과 제안을 주셔서 리차드 H​​. Masland 및 Akimichi 가네코.

<ol><li> 한천 블록 (30 MG / ML, 즉 3%, 증류수로)를 준비합니다. 용해와 전자렌지를 최초의 유리 그릇에 그것을 부어. 4 O C.에 냉장고에 음식 보관 팁 : 한천 블록이 사전에 준비되어야합니다. </li><li> 준비 낮은 온도 용해 아가로 오스 겔 (중간 25 MG / ML, 즉 2.5 %). 그것을 디졸브, 전자렌지. 35 O C.에있는 온수 욕조에 보관 팁 : 아가로 오스 겔이 너무 섹시하지 보관. 너무는 뜨겁다면, 그것은 확정되는 데 시간이 오래 걸립니다, 그리고 젤은 망막을 죽일 수 있습니다 . </li><li> 동물, 명백히하다에게 안구를 안락사와 세안 컵으로 엽니다. 유리체 유머를 녹일 수, 열린 세안 컵이있다면 반드시, 매체 hyaluronidase 10 분 (0.07 MG / ML)에 절어있다. </li><li> 블록 (예, 마우스 망막에 대한 1cm가 X 1.5 cm)에 한천을 잘라 굳게 인스턴트 접착제에 의해 vibratome 무대에 첨부하여 보내드립니다. 한천 블록이 젖었 유지. </li><li> 기계에서 5 정도에서 칼날을 놓습니다. 부드럽게 약의 속도에 한천 블록의 상단 표면을 잘라. 50 μm의 / 초, 주파수. 50 Hz에서. 팁 : 한천 블록의 상단 표면이 완전히 부드럽고 평평한되었는지 확인하십시오. 몇 가지 상처는 (적어도 3 회) 한천 블록의 부드럽고 평평한 표면을 필요 수 있습니다. </li><li> hyaluronidase없는 매체로 세안 컵을 씻어. 안료 상피에서 망막을 분리하여 필터 종이에 photoreceptor 쪽을 놓으십시오. 단단히 필터 종이에 망막을 첨부하려면 필터 종이에 여러 번 망막을 진공. 팁 : 망막이 완전히 평면 필터 종이에 첨부되어 있는지 확인합니다. 이 필터 종이에 망막을 넣어 후, 망막 (Fig.2A)를 둘러싼 초과 필터 종이를 잘라. 초과 여과지는 종종 망막의 해당 계층에 갈의 칼날을 방지합니다. </li><li> 부드럽게 한천 블록에있는 여분의 매체를 쓸어 조심스럽게 한천 블록에 필터 종이에 망막을 놓으십시오. 단단히 한천 블록에 여과지를 삽입하려면 필터 용지의 가장자리에 즉시 접착제를 첨부해 주시기 바랍니다. 팁 : unexpectable 균열을 방지하려면, 한천 블록의 상단 표면에 가능한 전면으로 망막를 놓습니다. </li><li> 부드럽게 망막을 통해 낮은 온도 용해 아가로 오스 젤 잔. 1 분 기다린 후 부드럽게 아래로 냉각로에있는 매체를 부어. 아가로 오스 겔은 신속하게 확정됩니다. 팁 : 너무 뜨거운 아가로 오스 겔은 망막을 죽이면. </li><li> 한천 블록의 상단 표면 위의 여러 수백 μm의에 칼날 위치를 허용하고 부드럽게 경화 아가로 오스 겔의 상단을 잘라. </li><li> 블레이드 위치를 내려. 내부 핵 계층 (한천 블록의 상단 표면 위 200 사이 250 μm의 약)의 근위 분의 수준에서 망막을 잘라요. 팁 : 블레이드 너무 많아 속도는 심각한 망막 손상을 것입니다. 약. 50 μm의 / 초 (50 Hz에서)이 적합합니다. </li><li> amacrine 세포가 처형될 위기에 다운됩니다 확인할 수 있습니다. 조심스럽게 경화 아가로 오스 겔와 함께 망막의 슬라이스를 선택하고 챔버 또는 접시에 얼굴까지 그것을 놓으십시오. 팁 : 마우스 망막은 작은과 물결 모양이기 때문에, 그것이 적절한 계층에서 완벽한 수평 슬라이스를 얻을 어렵습니다. &quot;경사&quot;슬라이스는 망막의 상대 평면 슬라이스를 얻을 다른 방법 수 있습니다. 경사 슬라이스를 얻으려면, 필터 종이와 같은 Fig.2B 같이 한천 블록에있는 망막 사이에 또 다른 필터 종이를 넣어. 당신은 완벽한 수평 단면을 기대할 수는 없지만, amacrine 세포의 소마는 조각의 표면에 일부 지역은 항상있다. </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>

모든 실험 프로토콜은 동물의 사용을위한 건강 지침 국립 연구소에 따라 실시되었다.

망막의 수평 슬라이스 준비의 여러 가지 중요한 장점이 있습니다. 첫째, 같은 amacrine 세포와 수평 세포와 같은 가로 dendrites를 확장하고 세포의 형태가 보존됩니다. 그것은 그것이 생리적 속성과 세포의 형태를 비교하는 것은 매우 중요하므로, 망막에 amacrine 세포의 20 종류 (맥닐 및 Masland, 1998) 이상이있다는 것을 보여왔다. 둘째, 전체 마운트 망막에 glial 세포의 endfeets의 광범위한 보험 amacrine 세포 패?...

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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

전통적으로 수직 슬라이스와 망막의 전체 마운트 준비는 망막 회로의 기능을 연구하는 데 사용되었습니다. 그러나, amacrine 세포로 망막 뉴런의 많은 세포의 형태가 심각하게 수직 조각에 손상 있어야되도록 가로 그들의 dendrites를 확장합니다. 전체 마운트 준비, 특히 패치 - 클램프 녹음을 위해 중간 계층에있는 망막 뉴런 인해 glial 세포 (뮬러 세포) 님의 endfeets의 광범위한 범위에 쉽게 액세스할 수 없습니다. 여기, 우리는 망막 손상 뉴런의 돌기 형태를 보존하는 방법을 깔끔히 소설을 설명합니다. 망막은 저온 용융 아가로 오스 겔에 포함된 이후 슬라이스는 vibratome 기계를 사용하여 망막의 내부 계층에서 수평으로 만들어졌습니다. 망막의 수평 슬라이스 준비에, 우리는 패치 - 클램프 녹음, 칼슘 이미징 기술, immunocytochemistry, RT - PCR 및 단일 세포를 사용하여, 그들의 형태와 비교하여 망막 뉴런의 기능을 공부했습니다.

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

Horizontal Slice Preparation of the Retina

전통적으로 수직 슬라이스와 망막의 전체 마운트 준비는 망막 회로의 기능을 연구하는 데 사용되었습니다. 여기, 우리는 망막 손상 뉴런의 돌기 형태를 보존하는 방법을 깔끔히 소설을 설명합니다.

망막의 수평 슬라이스 준비

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 ...

horizontal-slice-preparation-of-the-retina

Research

JoVE Journal

Biology

15.7K 조회수.  Dalhousie University. 전통적으로 수직 슬라이스와 망막의 전체 마운트 준비는 망막 회로의 기능을 연구하는 데 사용되었습니다. 여기, 우리는 망막 손상 뉴런의 돌기 형태를 보존하는 방법을 깔끔히 소설을 설명합니다.

비디오: 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.

마우스에 대한 진한 / 라이트 전환 테스트

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, ...

연구

생물학

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.

어른 토끼 망막의 Organotypic 문화

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.

Immunohistochemistry에 대한 슬라이스 준비 씬 Sectioning

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. 

E18 랫 두뇌의 Organotypic 슬라이스 문화

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.

진 건 글머리 기호 및 슬라이스 문화 뉴런의 Biolistic Transfection의 준비

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.

라이트의 전체 세포 레코딩은 망막 통에 흥분성의 시냅틱 전류를 Evoked

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.

Proteomic 분석을위한 마우스 유리체 및 망막의 내장 적출

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.

Morpholinos의 생체내의 Electroporation에서 성인 Zebrafish의 망막에

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.

배아 칙의 망막에서 비켜 Electroporation에

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.