Os autores gostariam de agradecer ao Dr. Louis Reichardt para os ratos TrkA taulacZ e Dr. Annette Markus para discussão perspicaz e sugestões. Este trabalho foi apoiado por fundos de inicialização da Fundação Burke, bem como Fundação Whitehall bolsa de investigação 2010-08-61, uma bolsa de investigação Wings for Life Foundation (WFL-US-028/14), conceder ZB1-1102-1 do Christopher e Dana Reeve Foundation, e concede 1R01EY022409 e 3R01EY022409-01S1 do National Eye Institute, a JZ. KJo é um companheiro Goldsmith.

NOTA: Todos os procedimentos em conformidade com o Manual do NIH para o uso e cuidados de animais de laboratório. O protocolo animal foi aprovada pelo IACUC no Weill Cornell Medical College. 1. Preparação do Tecido <ol><li> Eutanásia fêmeas cronometrado com a gravidez por deslocamento cervical 15. Dissecar embrionárias E16 - E18 embriões de fêmeas cronometrado com a gravidez e embriões lugar individualmente em poços de uma de 6 poços prato, preenchidas com solução salina fosfato tamponada (PBS). </li><li> Enxágüe embriões em PBS frio. </li><li> Retirar uma pequena porção da membrana amniótica do embrião e no lugar de uma solução de proteinase K pré-preparadas para a genotipagem subsequente. Alternativamente, se a membrana amniótica é perdida, remover uma pequena porção da ponta da cauda do embrião e colocá-lo em solução de proteinase K </li><li> Picar furos na pele ao redor do embrião usando pinos de insetos (pelo menos 100 de cada lado). </li><li> Remover PBS e lentamente adicionar fresco 2% paraformaldeído (PFA),pH 7,4, para o bem. </li><li> Agite suavemente durante 2 horas a 4 ° C. </li><li> Lavar embriões duas vezes em PBS e armazenar em PBS a 4 ° C até a coloração. </li></ol> 2. A genotipagem <ol><li> Mergulhe membranas amnióticas ou rabos de embriões em proteinase mistura K de acordo com as instruções do fabricante. </li><li> Incubar a 56 ° C durante 10 min. </li><li> Extrai-se o ADN utilizando um kit de purificação de ADN comercialmente disponível. </li><li> Utilizar 0,5 mL do total de 200 uL de solução de ADN genómico purificado, combiná-la com 0,5 ul de cada um dos iniciadores com sentido e anti-sentido específico para o gene (10 uM), 2 ul de dNTP Mix (2,5 mM de dATP, dCTP, dGTP e dTTP) , 0,2 jil de polimerase Taq (5 U / uL) e 2 ul de tampão de PCR (10x), adicionar H 2 O para um volume total de 20 ul. NOTA: As sequências dos iniciadores são como se segue (Tabela 1): TrkA: TrkA_F: GCG GGC GCC GCC GCG ATG, TrkA_R: GAA GCC GCC TGC GCG GCT CTG CCA GGG TG; PCR fragmento comprimento: 400 pares de bases (pb). Bax: In5R: GTT GAC CAG AGT GGC GTA GG, Ex5F: TGA TCA GAA CCA TCA TG, NeoR: CCG CTT CCA TTG CTC AGC GG; Comprimentos fragmento de PCR: tipo selvagem, 304 pb; Bax null, 507 pb. TauLacZ: lacZup: ACA ACG TCG TGA GTG GGA AAA, lacZdown: ATC AAC ATT AAA TGT GAG CGA G; PCR de comprimento de fragmentos: 360 pb. </li><li> Executar uma PCR com o seguinte programa para TrkA: passo 1: 1 min a 95 ° C, passo 2: 10 seg a 95 ° C, passo 3: 20 seg a 68 ° C, passo 4: 30 segundos a 72 ° C. Repita os passos 2-4 para 40 ciclos. <ol><li> Para LacZ e Bax, executar um PCR com o seguinte programa: passo 1: 1 min a 95 ° C, passo 2: 10 seg a 95 ° C, passo 3: 1 min a 54 ° C, passo 4: 1 min a 72 ° C, repita os passos 2-4 para 35 ciclos. </li></ol></li><li> Executar amostras de PCR num gel de agarose a 2% a 100 V, em tampão 1 x Tris-acetato-EDTA durante 20 min, com uma pista de escada de ADN para avaliar comprimentos de fragmentos. </li><li> Analise embriões para a presença do tipo selvagem de TrkA, TRKA-tauLacZ e Bax alelos nulos. Os genótipos de embriões experimentais são TrkA taulacZ / WT: Bax - / - e TrkA taulacZ / taulacZ: Bax - / -, dependendo das perguntas experimentais. Os embriões sem o repórter tauLacZ será negativo para a coloração axonal e pode, por conseguinte, servem como controlos negativos para calibrar o processo de coloração. </li></ol> 3. Embryo Coloração <ol><li> Usar um kit comercial para a coloração lacZ com algumas modificações como descrito em baixo nas secções 3,2-3,8. Aqui, use o kit Millipore. </li><li> Imergir embriões em Tampão A durante pelo menos 1 hora, sacudindo suavemente, à temperatura ambiente. </li><li> Imergir directamente embriões em tampão B durante pelo menos 15 min, agitando suavemente à temperatura ambiente. </li><li> Enquanto os embriões são em tampão A / B, pré-solução quente base de tecido a 37 ° C. Utilize 5 ml de solução de base Tissue por embrião. </li><li> Enquanto os embriões são em Tampão A / B, preparar fresco 5-brOmo 4- cloro-3- indolilo α-D-galactopiranósido (X-gal) a uma concentração de 40 mg / ml em 100% de dimetil sulfóxido (DMSO). </li><li> Dilui-se com X-gal para a solução de base de tecido pré-aquecido a uma concentração de 1 mg / ml e adicionam-se 5 ml de X-gal / mistura base de tecido para um frasco de cintilação de vidro. </li><li> Transferir os embriões para um frasco de cintilação de vidro contendo mistura de base de X-gal / Tissue. Selar tampa com Parafilm e incubar a 37 ° C durante a noite, verificando-se a presença de corante azul. </li><li> Postfix com embriões frescos PFA 4%, pH 7.4. Agitar suavemente durante 4 horas a 4 ° C. </li><li> Enxágüe embriões duas vezes em PBS frio. </li></ol> 4. Tissue Desidratação e Clearing <ol><li> Colocar em embriões de 50% de metanol / 50% de mistura de PBS durante 30 min à temperatura ambiente, agitando em frascos de vidro. </li><li> Continuar a desidratar em 75% de metanol / 25% de PBS durante 30 min à temperatura ambiente, agitando em frascos de vidro. </li><li> Continuar a desidratar em 90% de metanol / 10% PBS durante 30 min à temperatura ambiente, agitando em frascos de vidro. </li><li> Continuar a desidratar em metanol a 100% durante 30 min (2x), à temperatura ambiente, agitando em frascos de vidro. </li><li> Enquanto os embriões são desidratação, preparar uma mistura 1: 1 (v: v) de uma mistura de álcool de benzoílo e benzoílo benzoato. NOTA: álcool benzoílo e benzoílo benzoato é tóxico. </li><li> Imergir embriões no 1: 1 (v: v) de uma mistura de metanol a 100%: 100% BABB durante 15 min. Usar apenas porque o vidro BABB irá dissolver plástico. </li><li> Mergulhe embriões em 100% BABB para limpar completamente o tecido e visualizar coloração X-gal no embrião desmarcada. NOTA: A imagem o mais rapidamente possível, porque a mancha X-gal vai desaparecer ao longo do tempo. </li></ol> 5. Monte todo Imagem <ol><li> Estabilizar o embrião, imerso em 100% BABB, nos ângulos adequados para fotografia usando uma pinça de metal. Iluminar usando uma combinação de up-luz e sob luz luz fontes. </li><li> Adquirir imagens com um microscópio de dissecaçãoequipado com uma câmera digital. Tire várias exposições a campos brilhantes em cinco planos focais sucessivas 0,5 milímetros de distância ao longo do eixo Z. Os tempos de exposição e f-stops podem variar de acordo com a iluminação e câmera especificações e precisam ser otimizados para cada configuração. </li><li> Use um software de processamento de imagem padrão para processar imagens de sobreposição e gerar fotomontagens. </li></ol>

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The authors declare that there are no competing interests.

O processo de coloração X-gal acima descrito de ratos embrionárias TrkA taulacZ permite a visualização rápida e detalhada de projeções axônio de longa distância no embrião fixa intacta. Por causa do fundo nulo Bax estes ratos permitir a sondagem de mecanismos de sinalização que podem contribuir para o crescimento do axônio e sobrevivência neuronal. O cruzamento com camundongos transgênicos ou knockout de interesse permite uma avaliação abrangente de fenótipos axonal e pode...

Estabelecimento de redes neuronais precisas é um processo de desenvolvimento complexo essencial para a funcionalidade do sistema nervoso. Distúrbio neste processo leva à disfunção neuronal que tem sido implicado em doenças neurológicas humanas 1-3. Para estudar os mecanismos moleculares subjacentes de crescimento do axônio e inervação alvo em mamíferos, nós desenvolvemos um protocolo para visualizar as trajetórias axonal de TrkA-expressando neurônios sensoriais usando uma combinação de duas linhas de rato geneticamente modificados. TrkA é um receptor para o factor de crescimento dos nervos NGF e é um marcador funcional de neurónios sensoriais nociceptivos 4. TrkA é altamente expressa em neurónios nociceptivos durante o desenvolvimento precoce e medeia a sobrevivência de neurónios dependente de NGF, o crescimento axonal, arborização e alvo de inervação 5-9. Em ratinhos TrkA taulacZ, o gene de TrkA tipo selvagem é substituído por uma expressão de c taulacZassette 10, de tal modo que a morfologia axonal de neurónios TrkA-positivos putativos pode ser visualizado por β-gal (X-gal) 11 coloração. Usando uma linha de TrkA taulacZ / WT heterozigótico, pode-se examinar os factores que podem regular ou interferir com o desenvolvimento de projecções aferentes sensoriais in vivo. Além disso, a expressão de TrkA está ausente em ratinhos homozigóticos TrkA taulacZ / taulacZ, que pode, portanto, ser utilizados para avaliar a promoção do crescimento axonal na ausência de mecanismos de sinalização NGF / TrkA. Uma vez que os neurónios nociceptivos dependem sinalização NGF / TrkA não só para o crescimento axonal, mas também para a sobrevivência, que empregam uma outra linha de ratinhos, a que falta o gene de pró-apoptótica Bax, para inibir a apoptose em neurónios DRG embrionárias, salvando-los de morte celular que é de outra maneira observada na ausência de sinalização TrkA. O Bax - / - fundo 12 permite, assim, para o molecudissecção lar de vias de sinalização que afectam especificamente 7-9,13-15 crescimento axonal. Em TrkA - / -: Bax - / - ratos, neurónios DRG sobreviver, mas inervação aferente sensorial na pele é completamente abolida 14,15. Podemos ativar seletivamente as vias de sinalização para determinar suas respectivas contribuições para o desenvolvimento de projeções axônio. A utilidade deste método é que ele permite a avaliação de mudanças na fenótipos crescimento axonal quando diferentes modificações genéticas são criados para a TrkA taulacZ / taulacZ: Bax - / - ou TrkA taulacZ / WT: Bax - / - fundos.

<table border="0" cellpadding="0" cellspacing="0" width="465">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;"></td>
			<td style="width:127px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PFA</td>
			<td>Sigma-Aldrich</td>
			<td>P6418</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Life Tech</td>
			<td>10010-023</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue Rinse Solution A</td>
			<td>Millipore</td>
			<td>BG-6-B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue Rinse Solution B</td>
			<td>Millipore</td>
			<td>BG-7-B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue Stain Base Solution</td>
			<td>Millipore</td>
			<td>BG-8-C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">X-gal&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>B4252</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass scintiallation vial</td>
			<td>Kimble Chase</td>
			<td>74500-20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator</td>
			<td>Labline</td>
			<td>Model 120</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insect pins</td>
			<td>FST</td>
			<td>26000-30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 well dish</td>
			<td>USA Scientific</td>
			<td>CC7672-7506</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primers</td>
			<td>IDT</td>
			<td>custom DNA primers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Takara dNTP mixture</td>
			<td>Takara</td>
			<td>4030</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Takara LA buffer</td>
			<td>Takara</td>
			<td>RR002A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Takara LA Taq</td>
			<td>Takara</td>
			<td>RR002A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR machine</td>
			<td>Bio-Rad&#160;</td>
			<td>DNA Engine Dyad</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benzyl alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>B-1042</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benzyl benzoate</td>
			<td>Sigma-Aldrich</td>
			<td>B-6630</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting microscope</td>
			<td>Leica</td>
			<td>M205A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Camera</td>
			<td>Leica</td>
			<td>DFC310FX</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ring light&#160;</td>
			<td>Leica&#160;</td>
			<td>MEB110</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Photoshop</td>
			<td>Adobe</td>
			<td>Photoshop 4.0</td>
		</tr>
	</tbody>
</table>

whole-mount imaging of mouse embryo sensory axon projections

A visualização das projeções neuronais de corpo inteiro em embriões é essencial para ganhar uma compreensão de como mamíferos redes neuronais desenvolver. Aqui nós descrevemos um método para rotular in situ um subconjunto do gânglio da raiz dorsal (DRG) projeções axônio para avaliar suas características fenotípicas usando várias linhas de rato geneticamente manipulados. Os neurônios TrkA-positivos são neurônios nociceptores, dedicados à transmissão de sinais de dor. Nós utilizamos uma linha rato TrkA taulacZ para rotular as trajetórias de todos os axônios periféricos TrkA-positivos no embrião intacto mouse. Nós produzir ainda mais a linha TrkA taulacZ sobre um fundo nulo Bax, que essencialmente suprime a apoptose neuronal, a fim de avaliar as questões relacionadas com o crescimento, independentemente de possíveis efeitos das manipulações genéticas na sobrevivência neuronal. Posteriormente, os ratos geneticamente modificados, de interesse são criados com o TrkA TAULACZ / Bax linha nula e está então pronto para o estudo utilizando as técnicas aqui descritas. Esta apresentação inclui informações detalhadas sobre os planos de melhoramento do rato, genotipagem no momento da dissecção, a preparação do tecido, a coloração e de compensação para permitir a visualização de todo o comprimento trajetórias axonal em preparação todo o monte.

Os genótipos de TrkA WT / taulacZ: Bax - / - e TrkA taulacZ / taulacZ: Bax - / - embriões pode ser determinada sem ambiguidades por genotipagem de PCR padrão (Figura 1). X-gal de coloração exibe detalhado mandris axonal periféricos por via subcutânea em embriões coradas convencionalmente (Figuras 2, 3A), e em todo o embrião após clearing tecido (Figuras 3b, 4). Temos produzido a TrkA <sup...

Watch this Scientific Journal Video about Whole-mount Imaging of Mouse Embryo Sensory Axon Projections at JoVE.com

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

Todo-mount imagem de embrião de rato Projeções Sensorial Axon

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

whole-mount-imaging-of-mouse-embryo-sensory-axon-projections

Research

JoVE Journal

Neuroscience

10.0K visualizações.  Weill Medical College of Cornell University. We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

Vídeo: Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

Visualização do Sistema Nervoso embrionárias no Whole-mount Drosophila Os embriões

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

Neurociência

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

Múltiplas mouse-Imagem por Ressonância Magnética neuroanatômicos

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

Dissecação de um olho do rato para um monte inteiro do epitélio pigmentar da retina

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Reprodutível mouse esmagamento do nervo ciático e subsequente avaliação de Regeneração por Análise Whole Mount muscular

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

Whole Mount coloração de imunofluorescência do Rato Retina Neonatal para investigar angiogênese<em&gt; In vivo</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Monte todo imunomarcação de Neurônios Receptores Olfatórios no<em&gt; Drosophila</em&gt; Antena

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel cell clusters), sebaceous glands, nerves and nerve endings, and capillaries. The spatial arrangement of these structures is tightly controlled on a microscopic scale - as seen, for example, in the orderly arrangement of cell types within a single hair follicle - and on a macroscopic scale - as seen by the nearly identical orientations of thousands of hair follicles within a local region of skin. Visualizing these structures without physically sectioning the skin is possible because of the 2-dimensional geometry of this organ. In this protocol, we show that mouse skin can be dissected, fixed, permeabilized, stained, and clarified as an intact two dimensional object, a flat mount. The protocol allows for easy visualization of skin structures in their entirety through the full thickness of large areas of skin by optical sectioning and reconstruction. Images of these structures can also be integrated with information about position and orientation relative to the body axes.

Mammalian skin contains a diverse array of structures - such as hair follicles and nerve endings - that exhibit distinctive patterns of spatial organization. Analyzing skin as a flat mount takes advantage of the 2-dimensional geometry of this tissue to produce full-thickness high-resolution images of skin structures.

Plano Monte imagem de Rato pele e sua aplicação à análise do folículo piloso Padronização e Sensorial Axon Morfologia

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel ...

Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single dendrite to the surface of the epithelium, ending in a structure called dendritic knob. Cilia emanate from this knob into the mucus covering the epithelial surface. The proteins of the olfactory signal transduction cascade are mainly localized in the ciliary membrane, being in direct contact with volatile substances in the environment. For a detailed understanding of olfactory signal transduction, one important aspect is the exact morphological analysis of signaling protein distribution. Using light microscopical approaches in conventional cryosections, protein localization in olfactory cilia is difficult to determine due to the density of ciliary structures. To overcome this problem, we optimized an approach for whole mount labeling of cilia, leading to improved visualization of their morphology and the distribution of signaling proteins. We demonstrate the power of this approach by comparing whole mount and conventional cryosection labeling of Kirrel2. This axon-guidance adhesion molecule is known to localize in a subset of sensory neurons and their axons in an activity-dependent manner. Whole mount cilia labeling revealed an additional and novel picture of the localization of this protein.

Cilia of olfactory sensory neurons contain proteins of the signal transduction cascade, but a detailed spatial analysis of their distribution is difficult in cryosections. We describe here an optimized approach for whole mount labeling and en face visualization of ciliary proteins.

Monte todo Rotulagem de Cilia na Olfativo Sistema de Ratos Principal

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

Imagem Ca<sup&gt; 2+</sup&gt; Dynamics nos terminais Cone fotorreceptores Axon do mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

Monte todo Dissecção e Imunofluorescência do mouse cóclea Adulto

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

Todo-mount imagem de embrião de rato Projeções Sensorial Axon

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