Agradecemos aos membros do Laboratório de Marder. Agradecemos aos nossos conselheiro, Dr. Eve Marder, por seu apoio e encorajamento. Esta pesquisa foi apoiada pelo Instituto Nacional de Distúrbios Neurológicos e Derrame Grants NS-NS-17813 e 058110.

1. Dissecação bruta <ol><li> Coloque um caranguejo no gelo por 30 minutos para maçante os sistemas sensoriais e motoras do animal. Rodeiam por todos os lados com gelo. Devem ser usadas luvas ao manusear o caranguejo. </li><li> Prepare-se para a dissecção macroscópica fazendo o layout da pan dissecção e as ferramentas que você irá usar. Rongeurs, osso de corte de tesoura, tesoura pequena, pinça dentada, uma espátula com uma borda afilada, um prato Sylgard revestido preto, e pinos de insetos são necessários. </li><li> Remova o caranguejo do balde de gelo por sua perna para evitar que ele beliscar e colocá-lo na panela de dissecação. </li><li> Remova as garras primeiro torcendo cada uma para dentro. Em seguida, retire cada perna usando o mesmo movimento de torção. </li><li> Usando o fórceps, remova a maxila cobrindo a boca. Também remover o maxillule macio. </li><li> Usando o fórceps de novo, começar a partir da extremidade lateral posterior da carapaça e obter uma boa aderência na borda do mesmo. Reprimir com o fórceps e quebrar aquele pedaço de carapaça off. Certifique-se de remover o suficiente da carapaça para permitir a espátula para entrar na abertura. Continue removendo a borda da carapaça até os olhos e repita do outro lado. </li><li> Com o lado cônico da espátula, raspe o tecido mole interior longe do telhado da carapaça dorsal. Mantenha a espátula em contato com a carapaça para evitar danificar o STNS. Usar o fórceps de romper com as peças da carapaça que foram separados do tecido. Também separar alguns dos tecidos moles da carapaça ventral certificando-se de separar todos os músculos adutores prender o tecido para a carapaça. </li><li> Romper a carapaça dorsal, tanto posterior como o coração, onde existem dois pequenos ossículos saindo da carapaça. Corte, tanto anterior quanto possível. Estabilizar a Cephalon (a &quot;cara&quot;) com um dedo enquanto rompendo as partes mais anterior da carapaça dorsal. </li><li> No lado ventral da carapaça, puxe a uma mandíbula de uma vez por ancorar a fórceps em uma mandíbula, distorcendo-os para fora, para desalojar a mandíbula do epistome em anexo, e puxando-o para longe. Dois ossículos tempo com os músculos devem ser anexados à mandíbula extraída se feito corretamente. </li><li> Separe afastado o tecido dos lados da Cephalon perto dos olhos usando a espátula. Dissecção com tesoura pequena, corte todo o tecido tão perto do cume de ossicular como camada por camada possível. </li><li> Resto do caranguejo contra a parede da panela dissecção e sustentá-lo acima prendendo-o no lugar (as garras podem ser usados ​​para isso). Corte o labrum (&quot;lábio superior&quot;) de distância da epistome tomando cuidado para não deixar qualquer tecido em anexo. Usando osso de corte de tesoura fazer dois cortes diagonais na carapaça ventral para remover o Cephalon. </li><li> Leve o labrum com uma pinça dentada e cortou o lábio (&quot;lábio inferior&quot;) de distância da ossicular no lado ventral. Em seguida, levante suavemente o lábio longe do restante da carapaça ventral. Use a tesoura pequena para separar a parte ventral do estômago a partir do tecido que reveste a carapaça. Quando o piloro ampolas são visíveis, corte o estômago longe do tecido de cada lado até que o estômago é extraído do caranguejo. </li><li> Ainda segurando o estômago pelo lábio com uma pinça, o resto do estômago contra a palma da mão e coloque soro fisiológico na boca. Isso expande o estômago e facilita o corte aberto. Corte a partir da abertura da boca para baixo entre o direito através do piloro ossicular entre os ampolas. Em seguida, corte na diagonal para baixo através dos dois ramos do miocárdio. Cortar as pontas dos três dentes dentro do estômago. </li><li> Coloque o estômago em um grande, preto, prato Sylgard revestido com o interior do estômago para baixo. Pino para baixo dos cinco cantos da preparação com os pinos de insetos e encher a placa com solução salina fria, mergulhando toda a preparação (ver figura 1). </li></ol> 2. Dissecação fina <ol><li> , Dissecção Micro-ferramentas são necessárias para a dissecção fina. Em particular, pinças e tesouras primavera são necessários, bem como um suporte de pino com uma agulha de tungstênio. A clara, Sylgard revestido prato, petri e alguns pinos de fio fino também deveria ter sido preparado. </li><li> Início com a menor ampliação, começar por fixar para baixo as abas perto do lábio que costumava ser a abertura da boca. Reorganizar o resto dos pinos para fazer a preparação tenso e pin quaisquer outras áreas necessárias. </li><li> Levante cuidadosamente a hipoderme viu que cobrem a parte inferior da preparação e corte-lo em toda deixando um pedaço de hipoderme com dois pontos brancos. </li><li> Descasque a amarelo, tecido, wispy membranosa perto da parte inferior da preparação. O mvns são fracamente acoplada à parte inferior deste tecido. Separe cuidadosamente a mvns a partir do tecido amarelo como é puxado para cima. Siga o tecido amarelo e corte ao lado do cérebro. </li><li> Acompanhar os processos de espessura sair do cérebro que se estendem laterally. Estes são os nervos commisural. Uma vez que o fim do nervo comissural é atingido, retire a massa esponjosa, tecido off-white que foi cortado do lado do cérebro. </li><li> Onde os íons e os filhos de satisfazer as CGs, corte o tecido muscular e para revelar o tamanho dos íons e filhos. </li><li> Retornar para o patch hipoderme manchado e cortá-lo. Isso deve deixar uma abertura na artéria em torno do STG. Cortar a abertura para separar os dois músculos que ladeiam o STG. Levante as extremidades destes músculos e corte logo abaixo deles para separá-los da artéria e expor o alns e AGNs. Separar os músculos de acompanhamento, tanto para cima como a cartilagem deitado sobre o stn. </li><li> Cortar o tecido arterial em torno do STG até onde o mvns, dgn, DVN e se encontram. Trabalhar as DVN ao LVNs e cortar o tecido, esbranquiçada delicada que cobre o LVNs. </li><li> Localize a psn sobre o ossicular perto da parte inferior da preparação. O psn pode ser usado para localizar o lvn ea junção dlvn. </li><li> Em seguida siga as dlvn para baixo para localizar o pyn e pdn. O pyn muitas vezes envolve mais a ampola pilórica à abertura. O pdn garfos fora do dlvn e está entre os ampola pilórica eo cardio músculos válvula pilórica. Deixe algum músculo ligado a um desses nervos, porque eles podem ser facilmente confundidos quando a preparação é transferido para o prato Sylgard claro. </li><li> Depois de todos os nervos foram descobertos, cortar todas as conexões entre os restantes STNS e os nervos do tecido e indesejados do estômago. Mover cuidadosamente o STNS longe do resto do estômago, cortando as ligações não atendidas. </li><li> Condição o prato Sylgard clara com a massa restante do tecido do estômago, esfregando-o sobre a superfície do Sylgard até o Sylgard já não se sente muito pegajosa ou seca. Sylgard é hidrofóbico e STNS irá aderir fortemente a ele se o prato Sylgard revestido não está condicionada desta forma. </li><li> Adicione um pouco de soro fisiológico frio para o prato. Agarre as extremidades superiores dos nervos comissural com uma pinça e trazer o STNS ao prato Sylgard claro. </li><li> Pin do STNS para baixo na Sylgard. Sever do cérebro dos nervos comissural e usar os pinos Minuten para garantir as quatro extremidades das engrenagens para baixo primeiro. Garantir que a preparação é do lado certo, verificando que o ivn está apontando para cima longe do Sylgard. O dgn deve sair ligeiramente abaixo da STG quando o STG é do lado direito para cima. Pin o resto do nervo termina-se com os pinos de arame fino. </li><li> Limpe qualquer músculo ou tecido restante do STNS. </li><li> Desheath o STG usando um suporte de pino e um pouco viciado, agulha de tungstênio fino,. Faça um pequeno furo em um canto da bainha STG longe do corpo celular e use o buraco para chegar entre as camadas de revestimento. Corte cuidadosamente uma ponta da bainha separados para expor o neuropil STG e corpos celulares. </li><li> Pin qualquer flaps da bainha para baixo para aumentar a acessibilidade para os corpos celulares e estabilizar o STG para gravações intracelular. Re-pin o resto do STNS garantir que cada nervo é tenso e bem espaçados para a gravação extracelular (ver figura 3). </li></ol> 3. Resultados <ol><li> Idealmente, todos os nervos devem estar livres de nicks e danos, particularmente os que vão ser gravados a partir. Nenhum dos nervos devem ser enrolados ou trançados. O STG deve estar intacto, com todas as células dispostas em uma formação barba ao redor do neurópilo. O STNS intacta é bilateral simétrica e se parece com um homúnculo com o LVNs como as pernas, o mvns como os braços e as extremidade anterior como chefe. </li></ol> Abreviaturas: <table border="1"><tbody><tr><td> STNS </td><td> Sistema nervoso Stomatogastric </td></tr><tr><td> STG </td><td> Gânglio Stomatogastric </td></tr><tr><td> CoG </td><td> Gânglio comissural </td></tr><tr><td> OG </td><td> Gânglio esofágico </td></tr><tr><td> mvn </td><td> Nervo mediano ventricular </td></tr><tr><td> íon </td><td> Esofágico inferior do nervo </td></tr><tr><td> filho </td><td> Nervo esofágico Superior </td></tr><tr><td> AGN </td><td> Nervo gástrica anterior </td></tr><tr><td> ALN </td><td> Nervo lateral anterior </td></tr><tr><td> dgn </td><td> Nervo dorsal gástrica </td></tr><tr><td> DVN </td><td> Nervo dorsal ventricular </td></tr><tr><td> lvn </td><td> Nervo ventricular laterais </td></tr><tr><td> psn </td><td> Nervo posterior do estômago </td></tr><tr><td> pyn </td><td> Nervo pilórica </td></tr><tr><td> pdn </td><td> Nervo dilatador pilórica </td></tr><tr><td> dlvn </td><td> Ramo dorsal do nervo ventricular laterais </td></tr><tr><td> ivn </td><td> Nervo ventricular inferior </td></tr></tbody></table><img src="/files/ftp_upload/1207/figure_1_90ppi.jpg" alt= &quot;Figura 1&quot; /&gt; Figura 1: Diagrama ilustrando o posicionamento aproximado da STNS no estômago antes da dissecção. <img src="/files/ftp_upload/1207/figure_2_90ppi.jpg" alt="Figura 2" /> Figura 2: Diagrama de vários músculos na parte inferior do estômago e uma sobreposição da STNS em verde claro. Cada tipo de célula no STG está listado com os músculos são conhecidos para inervar. Este mapa é útil quando o planejamento para dissecar um nervo que transporta um sinal de uma determinada célula (cortesia do laboratório Nadim). <img src="/files/ftp_upload/old/1207_2009_1_12_5/figure_3.jpg" alt="Figura 3" /> Figura 3: Diagrama esquemático do STNS com eletrodos intracelulares e extracelulares poços (de Marder e Bucher, 2007).

<ol>
	<li>Harris-Warrick, R. M., Marder, E., Selverston, A. I., Moulins, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Dynamic Biological Networks: The stomatogastric nervous system. , (1992).</li><li>Marder, E., Bucher, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+circuit+dynamics+using+the+stomatogastric+nervous+system+of+lobsters+and+crabs.">Understanding circuit dynamics using the stomatogastric nervous system of lobsters and crabs.</a> Annu. Rev. Physiol. 69, 291-316 (2007).</li><li>Maynard, D. M., Dando, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+the+stomatogastric+neuromuscular+system+in+Callinectes+sapidus,+Homarus+Americanus+and+Panulirus+argus+(Decapoda+crustacean).">The structure of the stomatogastric neuromuscular system in Callinectes sapidus, Homarus Americanus and Panulirus argus (Decapoda crustacean).</a> Philos. Trans. R. Soc. Lond. B Biol. Sci. 268 (892), 161-220 (1974).</li><li>Selverston, A. I., Russell, D. F., Miller, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Stomatogastric+Nervous+System:+Structure+and+function+of+a+small+neural+network.">The Stomatogastric Nervous System: Structure and function of a small neural network.</a> Prog. Neurobiology. 7, 215-290 (1976).</li></ol>

A dissecção STNS é o primeiro passo na realização de um experimento de eletrofisiologia ou imuno-histoquímica, ou para a obtenção de células para cultura de células. Independentemente da experiência pretendida, a dissecção macroscópica permanecerá inalterada. No entanto, a dissecção multa pode variar. É importante planejar com antecedência quais as células e que os nervos você vai gravar a partir de. A maioria dos neurônios no STG têm processos que inervam pelo menos um músculo do estômago (ver ...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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>Black Sylgard 170</td>
<td>Other</td>
<td>Dow Corning</td>
<td>170</td>
<td>Gross Dissection</td>
</tr>
<tr>
<td>Fine Scissors</td>
<td>Surgery</td>
<td>FST</td>
<td>14090-09</td>
<td>&nbsp;</td>
<tr>
<td>Insect Pins size 6</td>
<td>Surgery</td>
<td>FST</td>
<td>26000-65</td>
<td>&nbsp;</td>
<tr>
<td>Mayo Scissors</td>
<td>Surgery</td>
<td>FST</td>
<td>14110-17</td>
<td>&nbsp;</td>
<tr>
<td>Micro-Spatula</td>
<td>Surgery</td>
<td>Ward Sci.</td>
<td>15 V 4313</td>
<td>&nbsp;</td>
<tr>
<td>Rongeurs</td>
<td>Surgery</td>
<td>FST</td>
<td>16000-14</td>
<td>&nbsp;</td>
<tr>
<td>Tissue Forceps</td>
<td>Surgery</td>
<td>FST</td>
<td>11021-12</td>
<td>&nbsp;</td>
<tr>
<td>Fine Tungsten Wire (0.001 in. diameter)</td>
<td>Surgery</td>
<td>California Fine Wire</td>
<td>&nbsp;</td>
<td>Fine Dissection</td>
</tr>
<tr>
<td>Forceps #5</td>
<td>Surgery</td>
<td>FST</td>
<td>91150-20</td>
<td>&nbsp;</td>
<tr>
<td>Minuten Pins</td>
<td>Surgery</td>
<td>FST</td>
<td>26002-10</td>
<td>&nbsp;</td>
<tr>
<td>Moria Spring Scissors (7mm blades)</td>
<td>Surgery</td>
<td>FST</td>
<td>15370-52</td>
<td>&nbsp;</td>
<tr>
<td>Petri Dish (100x15mm)</td>
<td>Other</td>
<td>Fischer	</td>
<td>08-757-12</td>
<td>&nbsp;</td>
<tr>
<td>Pin Holder</td>
<td>Surgery</td>
<td>FST</td>
<td>26018-17</td>
<td>&nbsp;</td>
<tr>
<td>Super fine forceps #5SF</td>
<td>Surgery</td>
<td>FST</td>
<td>11252-00</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 182</td>
<td>Other</td>
<td>Dow Corning</td>
<td>182</td>
<td>&nbsp;</td>
<tr>
<td>Tungsten Needles</td>
<td>Surgery</td>
<td>FST</td>
<td>10130-05</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting Microscope</td>
<td>Microscope</td>
<td>Wild</td>
<td>MC3</td>
<td>Misc.</td>
</tr>
<tr>
<td>Jonah Crab (C. borealis)</td>
<td>Animal</td>
<td>Commercial Lobster</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
			<div>
						
		</div>

cancer borealis stomatogastric nervous system dissection

O gânglio stomatogastric (STG) é um excelente modelo para estudar interações celulares e de rede, porque contém um número relativamente pequeno de células (aproximadamente 25 em C. borealis), que estão bem caracterizadas. As células do STG exibem uma ampla gama de produtos e são responsáveis ​​pelas ações motoras do estômago. O estômago contém o moinho gástrico que quebra os alimentos, com três dentes internos, e do piloro que filtra o alimento antes de chegar ao intestino médio. O STG produz duas saídas rítmica para controlar o moinho gástrico e do piloro conhecidos como geradores de padrão central (CPG). Cada célula do STG pode participar em um ou ambos os ritmos. Estes CPG permitir o estudo de neuromodulação, a homeostase, a variabilidade celular e rede, desenvolvimento da rede, e recuperação da rede. A dissecção do sistema nervoso stomatogastric (STNS) do caranguejo Jonas (Cancer borealis) é feito em duas partes: a dissecção macroscópica e multa. Na dissecção bruto todo o estômago é dissecada do caranguejo. Durante a dissecção fina do STNS é extraído do estômago usando um microscópio de dissecção e micro-dissecção ferramentas (ver figura 1). O STNS inclui o STG, o gânglio esofágica (OG), e os gânglios comissural (CoG), bem como os nervos que inervam os músculos do estômago. Aqui, vamos mostrar como realizar uma dissecação completa do STNS em preparação para um experimento de eletrofisiologia, onde as células do STG seria gravado a partir intracelularmente e os nervos periféricos seria usado para gravações extracelular. A técnica adequada para encontrar os nervos desejado é mostrado, assim como nossa técnica de desheathing o gânglio para revelar o somata e neuropil.

Watch this Scientific Journal Video about Cancer Borealis Stomatogastric Nervous System Dissection at JoVE.com

Cancer Borealis Stomatogastric Nervous System Dissection

O sistema nervoso stomatogastric (STNS) do caranguejo Jonas (<em&gt; C. borealis</em&gt;) Pode ser usado para eletrofisiologia, imuno-histoquímica, e os estudos de cultura de células. A extração STNS é feito em duas partes: a dissecção macroscópica e multa.

Câncer Borealis Dissection Sistema Nervoso Stomatogastric

The stomatogastric ganglion (STG) is an excellent model for studying cellular and network interactions because it contains a relatively small number of ...

cancer-borealis-stomatogastric-nervous-system-dissection

Research

JoVE Journal

Biology

Cancer Borealis Stomatogastric Nervous System Dissection

12.5K visualizações.  Brandeis. O sistema nervoso stomatogastric (STNS) do caranguejo Jonas ( C. borealis) Pode ser usado para eletrofisiologia, imuno-histoquímica, e os estudos de cultura de células. A extração STNS é feito em duas partes: a dissecção macroscópica e multa.

Vídeo: Cancer Borealis Stomatogastric Nervous System Dissection

Dissection of Drosophila Ovaries

Dissecção da Drosophila Ovários

Biologia

Dissection of 6.5 dpc Mouse Embryos

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell interaction and signaling mechanisms that guide embryonic patterning. However, dissection of the mouse embryo from the decidua shortly after implantation can be a challenging procedure, and detailed step-by-step documentation of this process is lacking.

Here we demonstrate how post-implantation (6.5 dpc) embryos are isolated by first dissecting the uterus of a pregnant mouse (detection of the vaginal plug was designated day 0.5 poist coitum) and subsequently dissecting the embryo from maternal decidua. The dissection of Reichert's membrane is described as well as the removal of the ectoplacental cone.

Isolation of postimplantation-stage embryos allows one to study gene patterning and analyze cell-lineage decision making processes during embryonic development, but proper dissection of the early embryo can be challenging. This protocol describes a method for isolating early primitive-streak-stage embryos (~6.5 days post coitum [dpc]).

Dissecção de 6,5 embriões de camundongos dpc

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell ...

Isolation of Mononuclear Cells from the Central Nervous System of Rats with EAE

Whether studying an autoimmune disease directed to the central nervous system (CNS), such as experimental autoimmune encephalomyelitis (EAE, 1), or the immune response to an infection of the CNS, such as poliomyelitis, Lyme neuroborreliosis, or neurosyphilis, it is often necessary to isolate the CNS-infiltrating immune cells.
In this video-protocol we demonstrate how to isolate mononuclear cells (MNCs) from the CNS of a rat with EAE. The first step of this procedure requires a cardiac perfusion of the rodent with a saline solution to ensure that no blood remains in the blood vessels irrigating the CNS. Any blood contamination will artificially increase the number of apparent CNS-infiltrating MNCs and may alter the apparent composition of the immune infiltrate. We then demonstrate how to remove the brain and spinal cord of the rat for subsequent dilaceration to prepare a single-cell suspension. This suspension is separated on a two-layer Percoll gradient to isolate the MNCs. After washing, these cells are then ready to undergo any required procedure. 
Mononuclear cells isolated using this procedure are viable and can be used for electrophysiology, flow cytometry (FACS), or biochemistry. If the technique is performed under sterile conditions (using sterile instruments in a tissue culture hood) the cells can also be grown in tissue culture medium. A given cell population can be further purified using either magnetic separation procedures or a FACS.

In this video we demonstrate how to isolate mononuclear cells from the central nervous system of rats with experimental autoimmune encephalomyelitis.

Isolamento de células mononucleares do Sistema Nervoso Central de ratos com EAE

Whether studying an autoimmune disease directed to the central nervous system (CNS), such as experimental autoimmune encephalomyelitis (EAE, 1), or the ...

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

Drosophila larval Dissection MNJ

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.

Dissecção da Saccharomyces cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

Homarus Americanus Stomatogastric Nervous System Dissection

With the goal of understanding how nervous systems produce activity and respond to the environment, neuroscientists turn to model systems that exhibit the activity of interest and are accessible and amenable to experimental methods. The stomatogastric nervous system (STNS) of the American lobster (Homarus americanus; also know was the Atlantic or Maine lobster) has been established as a model system for studying rhythm generating networks and neuromodulation of networks. The STNS consists of 3 anterior ganglia (2 commissural ganglia and an oesophageal ganglion), containing modulatory neurons that project centrally to the stomatogastric ganglion (STG). The STG contains approximately 30 neurons that comprise two central pattern generating networks, the pyloric and gastric networks that underlie feeding behaviors in crustaceans1,2. While it is possible to study this system in vivo3, the STNS continues to produce its rhythmic activity when isolated in vitro. Physical isolation of the STNS in a dish allows for easy access to the somata in the ganglia for intracellular electrophysiological recordings and to the nerves of the STNS for extracellular recordings. Isolating the STNS is a two-part process. The first part, dissecting the stomach from the animal, is described in an accompanying video article4. In this video article, fine dissection techniques are used to isolate the STNS from the stomach. This procedure results in a nervous system preparation that is available for electrophysiological recordings.

Homarus Americanus Stomatogastric Nervous System Dissection

We describe the fine dissection of the stomatogastric nervous system from the stomach of the American lobster (Homarus americanus).

Homarus Americanus Stomatogastric Dissecção do Sistema Nervoso

With the goal of understanding how nervous systems produce activity and respond to the environment, neuroscientists turn to model systems that exhibit the ...

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

Dissecção de Órgãos do Zebrafish Adulto

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

Deficient Pms2, ERCC1, Ku86, CcOI in Field Defects During Progression to Colon Cancer

In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other malignancies of the same tissue type, often in a nearby location. Such field defects have been indicated in colon cancer. The molecular abnormalities that are responsible for a field defect in the colon should be detectable at high frequency in the histologically normal tissue surrounding a colonic adenocarcinoma or surrounding an adenoma with advanced neoplasia (well on the way to a colon cancer), but at low frequency in the colonic mucosa from patients without colonic neoplasia.
Using immunohistochemistry, entire crypts within 10 cm on each side of colonic adenocarcinomas or advanced colonic neoplasias were found to be frequently reduced or absent in expression for two DNA repair proteins, Pms2 and/or ERCC1. Pms2 is a dual role protein, active in DNA mismatch repair as well as needed in apoptosis of cells with excess DNA damage. ERCC1 is active in DNA nucleotide excision repair. The reduced or absent expression of both ERCC1 and Pms2 would create cells with both increased ability to survive (apoptosis resistance) and increased level of mutability. The reduced or absent expression of both ERCC1 and Pms2 is likely an early step in progression to colon cancer.
DNA repair gene Ku86 (active in DNA non-homologous end joining) and Cytochrome c Oxidase Subunit I (involved in apoptosis) had each been reported to be decreased in expression in mucosal areas close to colon cancers. However, immunohistochemical evaluation of their levels of expression showed only low to modest frequencies of crypts to be deficient in their expression in a field defect surrounding colon cancer or surrounding advanced colonic neoplasia.
We show, here, our method of evaluation of crypts for expression of ERCC1, Pms2, Ku86 and CcOI. We show that frequency of entire crypts deficient for Pms2 and ERCC1 is often as great as 70% to 95% in 20 cm long areas surrounding a colonic neoplasia, while frequency of crypts deficient in Ku86 has a median value of 2% and frequency of crypts deficient in CcOI has a median value of 16% in these areas. The entire colon is 150 cm long (about 5 feet) and has about 10 million crypts in its mucosal layer. The defect in Pms2 and ERCC1 surrounding a colon cancer thus may include 1 million crypts. It is from a defective crypt that colon cancer arises.

Reduced/absent expression of Pms2 and/or ERCC1 in entire crypts is a frequent event within 10 cm on each side of colonic adenocarcinomas, likely the basis of a field defect with high mutability and progression to cancer. Deficiency in Ku86 or CcOI is much less frequent in these field defects.

PMS2 deficiente, ERCC1, Ku86, CCOI Defeitos em campo durante a progressão para cancro do cólon

In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

Dissecção e coloração de Drosophila Larval Ovários

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

A BW Reporter System for Studying Receptor-Ligand Interactions

Interactions between receptors and ligands constitute a fundamental biological process. However, direct experiments with cells that express the native receptor and the ligand are challenging since the ligand of a specific receptor may be unknown and experimental procedures with the native ligand can be technically complicated. To address these obstacles, we describe a reporter system to detect the binding and activation of a specific receptor by a ligand of interest. In this reporter system, the extracellular domain of a specific receptor is conjugated to mouse CD3&#950; and this chimeric protein is then expressed in mouse BW cells. These transfected BW cells can then be incubated with different targets (e.g., cells or antibodies). Activation of a transfected receptor leads to the secretion of mouse interleukin-2 (mIL-2) which can be detected by enzyme-linked immunosorbent assay (ELISA). This reporter system has the advantages of being sensitive and specific to a single receptor. In addition, the activation level of a specific receptor can easily be quantified and can be used even in cases where the ligand of the receptor is unknown. This system has been implemented successfully in many of our studies to characterize receptor-ligand interactions. We have recently employed this system to study the activation of human Fc&#947; receptors (Fc&#947;Rs) by different monoclonal anti-CD20 antibodies in clinical use.

This protocol describes how to establish a reporter system that can be used to identify and quantify receptor-ligand interactions.

Um sistema BW repórter para estudar interações ligante-Receptor

Interactions between receptors and ligands constitute a fundamental biological process. However, direct experiments with cells that express the native ...