We thank the Advanced Microscopy Unit (School of Life Sciences, University of Nottingham) and the Histology team of MRC Human Reproductive Sciences Unit (Edinburgh), the Institute of Neuroscience at the ULB and the FNRS for help and support. This work was supported by MRC (MR/L001047/1 to A.D.J.).

Todos os procedimentos envolvidos em animais foram realizados de acordo com o comitê de ética da Universidade de Nottingham de. 1. Seleção de preparação do tecido apropriado para imunocoloração <ol><li> Gerar secção embebidos em parafina de embriões de tipo selvagem de ratinho CD1 e tecidos do cérebro adulto, conforme descrito anteriormente 25. Usar secções de tecido do cérebro fixadas com formaldeído a 4%, quer (FA) ou paraformaldeído a 4% (PFA) para o método de imunocoloração 25. NOTA: O uso de cortes de tecido embebidos em parafina requer de-depilação antes da marcação de anticorpos. Uma vez que o tratamento com 2-4 M de ácido clorídrico (HCl) empregada no protocolo para a desnaturação do DNA não é compatível com a maioria das estratégias de recuperação antigênica, recomendamos o uso de qualquer seções cryo- ou micrótomo para co-detecção de derivados de oxidação 5mC com proteína marcadores. </li></ol> 2. Desparafinação parafina tecido embebido secções &lt;<html> / P&gt; <ol><li> Em uma cabine de segurança classe running II, lavar a parafina secções de tecido embebidas numa jarra de Coplin cheio com xileno, 2 vezes para 10 minutos cada em temperatura ambiente. NOTA: É importante a utilização de xileno fresco como parafina remoção incompleta pode conduzir a padrões de coloração inconsistentes. </li><li> Depois da-cera, re-hidratar rapidamente as secções de tecido por lavagem consecutivamente em 95, 75, e 50% de etanol durante 10 minutos cada à temperatura ambiente. </li></ol> 3. Fixação e permeabilização de Cryo- e secções do micrótomo <ol><li> Fixar as secções de tecido re-hidratadas, colocando-os em qualquer gelo frio PFA a 4% ou 4% de FA durante 15 min à TA. </li><li> Remover o excesso de fixador por lavagem das secções em PBS (salina tamponada com fosfato) durante 5 min à TA. </li><li> Permeabilizar as secções de tecido, colocando num frasco Coplin preenchido com o PBX (0,5% de Triton X-100 em PBS) durante 30 min à TA. Retire o excesso de PBX por lavagem das secções em breve em PBT (0,01% de Tween 20 em PBS). </li></ol>jove_title &quot;&gt; 4. imunocoloração para oxi-5mC Derivativos <ol><li> Coloque as secções permeabilizadas em HCl 2 N durante 60 min à TA durante despurinação do DNA. Nota: Embora as concentrações mais elevadas de HCl (por exemplo, 4 N) conduz a uma desnaturação do DNA mais eficiente, que não são compatíveis para co-detecção de oxi-MCS com DAPI. </li><li> Coloque as secções em Tris-HCl a 10 (pH 8,5) durante 30 min à temperatura ambiente para neutralizar o HCl. Em alternativa, as secções lavar três vezes durante 5 minutos cada em PBS. Incubam-se as secções em PBT durante 5 min à TA. </li><li> Remova cuidadosamente o líquido da área circundante a secção de tecido, sem deixar que a secção de tecido secar completamente em qualquer etapa agitando suavemente o slide. Use uma caneta barreira hidrofóbica para cercar a seção sem tocar na seção. NOTA: A caneta barreira hidrofóbica reduz o volume de anticorpo necessário para manchar o tecido e pode permitir várias seções de ser marcadas com differenanticorpos t no mesmo slide. </li><li> Incubar secções em 100 ul de solução de bloqueio (albumina de soro bovino a 10% em PBS) durante 1 h à temperatura ambiente numa câmara húmida. NOTA: Ignorar este passo não afetaria a eficiência da coloração das bases de citosina modificadas. </li><li> Incubam-se as secções de tecido em 100 ul de uma diluição 1: 5000 de anticorpo monoclonal anti-5hmC e uma diluição 1: 1000 de anticorpos primários policlonais de coelho anti-5caC na solução durante 1 h à TA, o bloqueio numa câmara húmida. Alternativamente, realizar a incubação durante a noite a 4 ° C, se necessário. NOTA: Secções processados ​​sem anticorpos primários podem servir como controlos negativos adequados para o processo de coloração. As secções de 12,5 dias pós-embrionárias de murídeo enriquecidas em coitum cerebrais 5caC, 5FC e 5hmC 20 pode ser usado como controlo positivo. </li><li> Remover o excesso de anticorpos por lavagem das secções em um frasco Coplin preenchido com PBT três vezes durante 5 min cada em uma bobinajar lin à temperatura ambiente. NOTA: O aumento do volume de soluções de lavagem pode reduzir a coloração de fundo. </li><li> Remover o excesso de PBT e, se necessário, rodear as secções mais uma vez com a caneta barreira hidrofóbica como PBT conter detergente que pode enfraquecer a barreira hidrofóbica. </li><li> Fazer uma diluição 1: 400 de anticorpo conjugado com HRP de cabra anti-coelho e uma diluição 1: 400 de anticorpo-555 conjugado de burro anti-rato em solução de bloqueio. </li><li> Incubam-se as secções de tecido em 100 ul de mistura de anticorpo secundário a partir do passo 4.9 durante 1 hora à temperatura ambiente numa câmara húmida. </li><li> Lavam-se as secções de tecido em um frasco Coplin preenchido com PBT três vezes durante 5 minutos cada à temperatura ambiente. </li><li> Coloque as secções de tecido em 100 ul de uma diluição 1: 200 de tiramida no tampão de amplificação de sinal de tiramida durante 2 min à temperatura ambiente. </li><li> Imediatamente, remover a solução de tiramida excesso por lavagem das lâminas três vezes durante 5 min cada em PBT. </li><li> carefully remover o excesso de PBT e cobrir imediatamente as seções com uma gota de um meio de montagem (veja Lista de Materiais). </li><li> Delicadamente, coloque uma lamela sobre os cortes de tecido e selar imediatamente a lamela com unha polonês. </li><li> Armazenar as secções de tecidos a 4 ° C durante várias horas antes do exame microscópico. Examinar sob um microscópio de fluorescência a 405, 488 e / ou 555 nm (10 - ampliação de 40X). </li></ol>

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The authors declare no conflict of interest.

Embora a baixa abundância relatado de derivados de oxidação 5mC, 5FC e 5caC em alguns tecidos iria apresentar limitações significativas para um protocolo imunoquímica standard, a incorporação de anticorpos secundários conjugados com peroxidase permitiu a detecção dessas modificações citosina em tecidos e células fixas (Figura 2) . No entanto, o tempo de incubação óptimo com a solução tiramida devem ser optimizadas experimentalmente para cada lote individual de tiramida kit de amplificação de sinal em que uma relação linear entre a intensidade do sinal e a duração de amplificação de sinal com base tiramida é observada, para indicações referem-se Almeida et ai 2012 26. . Além disso, a relação sinal / fundo pode ser significativamente aumentada através da realização das lavagens em um frasco Coplin para permitir a remoção eficiente do excesso de anticorpos. Após a incubação com uma solução de tiramida, é importante para parar imediatamente a reacção por lavagem em solução de d PBTfundo ecrease coloração. É crítico para não permitir que as secções para secar a qualquer momento durante o procedimento. A eficiência de despurinação de ADN pode ser melhorada por se realizar a reacção a depurinação a 37 ° C. Enquanto utilizando HCl 4 N em vez de 2 N melhora a coloração das formas modificadas de 5mC utilizando HCl 4 N para a despurinação do DNA não iria permitir a co-coloração com DAPI, à medida que interage exclusivamente com o ADN de cadeia dupla. Embora esta técnica pode fornecer uma avaliação semi-quantitativa robusta das formas modificadas de bases de citosina onde detectável, ele não pode ser usado para avaliar os níveis absolutos de seus derivados de oxidação ou 5mC. Portanto, recomendamos a utilização de outros complementares, mas quantitativa aproxima 16, 19 -24. Desde a descoberta de derivados de oxidação 5mC em genomas de mamíferos, várias abordagens têm sido desenvolvidas para estudar os seus papéis biológicos 16, 19-24. Embora estes approaches pode ser valiosa na determinação dos níveis absolutos de derivados de oxidação 5mC, eles não fornecem informações relativas à sua distribuição espacial 20, 26. Temos utilizado com sucesso o método descrito aqui para mapear a distribuição espacial e localização de derivados de oxidação 5mC em diferentes tipos de células do desenvolvimento e cérebro adulto 20 Ao revelar sua distribuição espacial 20, esta técnica pode ser crucial para compreender as implicações biológicas e o destino de derivados oxidados de 5mC em vários contextos biológicos onde estes derivados modificados de 5mC podem ser detectados incluindo diferenciação celular, desenvolvimento e doença. Além disso, o método aqui descrito pode dar avaliação semi-quantitativa dos derivados oxidados de 5mC em diferentes tecidos, avaliando a cinética de reacção de peroxidase (que é proporcional à intensidade de coloração) em diferentes tempos de incubação com tyramide 26.

Metilação de bases de citosina no DNA (5mC) representa uma importante marca epigenética encontrado em vertebrados genomas associadas a transcrição silenciamento 1. 5mC está a ser introduzido e mantido pela ADN metiltransferases 2-5, e tem sido demonstrado que desempenham papéis importantes numa série de processos biológicos incluindo imprinting genômico, inactivação do cromossoma X, a diferenciação celular, e desenvolvimento de 3, 6. Em consequência, a interrupção de padrões de 5mC genómicas está associada com um número de doenças 7, 8-11. Apesar dos progressos na compreensão papel 5mC no desenvolvimento e na doença, ainda é permanecem largamente desconhecidos como esta marca está sendo removido no desenvolvimento e tecidos adultos. Vários mecanismos potenciais de desmetilação de ADN foram recentemente propostas incluindo mecanismos ativos e passivos desmetilação 12, 13, 14, 15. Descoberta dos produtos de 5mC oxidação sequencial mediadas por 1011 translocação enzymES (tet1 / 2/3), tais como 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5FC), e 5-carboxylcytosine (5caC) em eucariótica de ADN 16, 17, 18, ​​19 solicitado especulações se eles podem servir como intermediários de processos desmetilação de ADN ou marcas epigenéticas agir como estáveis ​​em seu próprio direito 13. Enquanto a demonstração de que o componente de base de reparo de excisão, glicosilase-DNA timina (TDG) pode ligar e remover tanto 5FC e 5caC de DNA 19, 20 sugere um papel para os derivados 5mC modificados numa desmetilação DNA ativo. Evidências recentes mostram que 5FC / 5caC pode modular a taxa de pontos de processabilidade RNA II para o potencial envolvimento destas marcas na regulação da transcrição 29. Devido a este potencial importância biológica das formas oxidadas de 5mC, uma variedade de técnicas bioquímicas e de anticorpos com base têm sido utilizados para estudar a sua distribuição genómico e conteúdo global 16, 19-24. Dado que a maioria de Tele vertebrado órgãos consistem em diferentes tipos de células e que a distribuição das bases de citosina modificadas é o tecido e tipo celular específico 16-18, 20, 23, 25-27, determinar a distribuição espacial dos derivados oxidados 5mC em diferentes tecidos torna-se um importante experimental tarefa necessária para revelar as suas funções biológicas. A maioria das abordagens bioquímicas e de anticorpos com base não fornecem qualquer informação espacial em relação à distribuição das formas modificadas de 5mC no tecido diferente e tipos de células. Em contraste, técnicas de imunoquímica base pode proporcionar uma ferramenta para a avaliação rápida da distribuição espacial e localização nuclear de 5mC 28 e os seus derivados oxidados 20. O que é dito, o relatado muito baixa abundância de 5FC (20 em cada 10 6 citosina) e 5caC (3 em cada 10 6 citosina) no genoma do rato 18 representa um desafio significativo para imunoquímica padrão. Aqui,descrevemos um método imunoquímico altamente sensível que proporciona robusta e uma rápida detecção da forma oxidada de citosina no tecido cerebral de mamífero. Com a incorporação de anticorpos secundários conjugados com peroxidase acoplada com um passo de amplificação de sinal, este método evita os desafios de detectar a quantidades muito baixas de 5FC e 5caC. Além disso, esta técnica pode ser utilizada para co-detectar as formas modificadas de citosina com marcadores específicos de linhagem, que complementa eficazmente outras abordagens em elucidar as funções biológicas destas marcas epigenética.

<table border="0" cellpadding="0" cellspacing="0" width="1281">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:388px;">Coplin jar</td>
			<td style="width:180px;">Cole-Parmer</td>
			<td style="width:119px;">UY-48585-30</td>
			<td style="width:595px;">Glass made</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Xylene</td>
			<td></td>
			<td></td>
			<td>Use only in Class II safety cabinet</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate buffer saline (PBS)</td>
			<td>Fisher Scientific</td>
			<td>12821680</td>
			<td>pPH 7.5, filtered before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4% paraformaldehyde (PFA)</td>
			<td>Sigma Aldrich</td>
			<td>P6148</td>
			<td>Once made can be stored at -20&#176;C in aliquotes for several months</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4% formaldehyde&#160; (FA)</td>
			<td>Sigma Aldrich</td>
			<td>F8775</td>
			<td>Once made can be stored at -20&#176;C in aliquotes for several months</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol, anhydrous denatured, histological grade</td>
			<td>Sigma Aldrich</td>
			<td>&#160;64-17-5&#160;&#160;</td>
			<td>95, 75, 50 % in PBS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.01 % Tween 20 in PBS</td>
			<td>Sigma Aldrich</td>
			<td>P9416</td>
			<td>PBT</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.5% Triton X-100 in PBS</td>
			<td>Sigma Aldrich</td>
			<td>X100</td>
			<td>PBX</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2 N HCl</td>
			<td>Sigma Aldrich</td>
			<td>71826</td>
			<td>caution extremely toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100 mM Tris-HCl&#160;</td>
			<td>Promega</td>
			<td>H5121</td>
			<td>PH adjusted to 8.5</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hydrophobic barrier pen</td>
			<td>Abcam</td>
			<td>ab2601</td>
			<td>for immunohistochemistry</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Slide moisture chamber</td>
			<td>Scientific Device Laboratory</td>
			<td>197-BL</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-5mC mouse antibody</td>
			<td>Diagenode</td>
			<td>C15200081</td>
			<td>monoclonal primary antibody</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-5hmC antibody</td>
			<td>Active Motif</td>
			<td>39791</td>
			<td>rabbit polyclonal primary antibody</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-5caC antibody</td>
			<td>Active Motif</td>
			<td>61225</td>
			<td>rabbit polyclonal primary antibody</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Peroxidase-conjugated anti-rabbit seconday antibody</td>
			<td>Dako</td>
			<td>K1497</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">555-congjuated goat anti-mouse antibody</td>
			<td>Molecular probes</td>
			<td>A-11005</td>
			<td>&#160;secondary antibody</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10% BSA</td>
			<td>Sigma Aldrich</td>
			<td>A9418</td>
			<td>Blocking solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">22x32mm Glass cover slips</td>
			<td>BDH</td>
			<td>406/0188/24</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tyramide Signal Amplification System</td>
			<td>Perkin Elmer</td>
			<td>NEL741001KT</td>
			<td>Other fluorochome conjugated seconday antibodies can be used for co-detection of oxi-5mC&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;Mounting Medium with DAPI</td>
			<td>Vector Labs</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Colourless nail polish</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">chicken polyclonal anti-GFAP polyclonal antibody</td>
			<td>Thermo Scientific</td>
			<td style="width:119px;">PA5-18598</td>
			<td>1:400 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-NeuN mouse monoclonal antibody</td>
			<td>Merck milipore</td>
			<td style="width:119px;">MAB377B</td>
			<td>1:400 dilution</td>
		</tr>
	</tbody>
</table>

detection of modified forms of cytosine using sensitive immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

Para determinar a distribuição de 5hmC em secções de tecido cerebral, foi realizado co-detecção desta modificação epigenética com um marcador para neurónios pós-mitóticos, NeuN, empregando anticorpo-5hmC anti comercial que interage especificamente com esta marca, mas não com outras formas de modificação citosina 20, 25. a análise imuno-histoquímica de distribuições 5hmC e 5caC no cérebro adulto revelou que, enquanto a coloração proeminente 5hmC co-localiza com as células positivas NeuN, células gliais NeuN-negativas possuem níveis mais baixos de 5hmC genómico (Figura 1) 20. Recentemente, mostrou que a oxidação 5mC Tet-dependente é operatório durante a especificação das linhagens de células-tronco neurais (NCCC) 20. Apesar de ser imunoquimicamente indetectável no NSC, 5FC e 5caC exibem imuno proeminente nas primeiras fases de diferenciação NSC no sentido neuronal uma<html> nd linhagens gliais. Ambas as marcas transitoriamente acumular concorrentemente com a aparência de marcadores de diferenciação neuronal e glial precoce 20. Para determinar a distribuição de 5caC na diferenciação NSC nós realizadas co-coloração deste traço com um marcador glial GFAP sobre as culturas fixadas de NSC aos 3 dias de indução da diferenciação glial. Ao contrário de NSC ou astrócitos maduros (dados não apresentados), observou-se sinal forte em 5caC relativamente grande proporção das células que expressam GFAP nestas culturas (Figura 2) 20. <img alt="figura 1" src="/files/ftp_upload/54416/54416fig1.jpg" /> Figura 1. Co-imunocoramento 5hmC (verde) com NeuN (vermelho) no tecido adulto Cérebro contrastadas com DAPI (azul). Os canais individuais ea exibição mesclada são mostrados. As barras de escala são de 25 uM.blank &quot;&gt; Clique aqui para ver uma versão maior desta figura. <img alt="Figura 2" src="/files/ftp_upload/54416/54416fig2.jpg" /> Figura 2. Co-imunocoramento 5caC com GFAP na cultura do NSC no dia 3 de glial diferenciação. Os canais individuais ea exibição mesclada são mostrados. Barras de escala são 20 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54416/54416fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>

Watch this Scientific Journal Video about Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry at JoVE.com

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

Detecção de formas modificadas da citosina Utilizando imunohistoquímica Sensível

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. ...

detection-modified-forms-cytosine-using-sensitive

Nanyang Technological University

Research

JoVE Journal

Biology

7.2K visualizações.  Université Libre de Bruxelles. Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

Vídeo: Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Thin Sectioning of Slice Preparations for Immunohistochemistry

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

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

Seccionamento fina de Preparações Slice para Imunohistoquímica

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

Biologia

Preventing the Spread of Malaria and Dengue Fever Using Genetically Modified Mosquitoes

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission.   Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced, as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed.

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission. Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed.

Impedir a propagação da malária e dengue por meio de mosquitos geneticamente modificados

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission.   Population ...

Immunohistochemistry: Paraffin Sections Using the Vectastain ABC Kit from Vector Labs

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific antibodies. There are two methods: the direct and indirect method. In this experiment, we will only describe the use of indirect IHC staining. Indirect IHC staining utilizes highly specific primary and biotin-conjugated secondary antibodies. Primary antibodies are utilized to discretely identify proteins of interest by binding to a specific epitope, while secondary antibodies subtract for non-specific background staining and amplify signal by forming complexes to the primary antibody. Slides can either be generated from frozen sections, or paraffin embedded sections mounted on glass slides. In this protocol, we discuss the preparation of paraffin-embedded sections by dewaxing, hydration using an alcohol gradient, heat induced antigen retrieval, and blocking of endogenous peroxidase activity and non-specific binding sites. Some sections are then stained with antibodies specific for T cell marker CD8 and while others are stained for tyrosine hydroxylase. The slides are subsequently treated with appropriate secondary antibodies conjugated to biotin, then developed utilizing avidin-conjugated horseradish peroxidase (HRP) with Diaminiobenzidine (DAB) as substrate. Following development, the slides are counterstained for contrast, and mounted under coverslips with permount. After adequate drying, these slides are then ready for imaging.

Imuno-histoquímica: Seções de parafina Usando o Kit de Vectastain ABC Vector Labs

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific ...

Immunohistochemistry on Paraffin Sections of Mouse Epidermis Using Fluorescent Antibodies

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, suprabasal, and stratum corneum layers. The precise localization within the epidermis of a particular protein lends clues toward its functional role within the epidermis. In this chapter, we describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory. Paraffin sections generally provide much better morphology, hence, superior results and photographs; however, not all antibodies will work with the harsh fixation and treatment involved in their processing. Therefore, the protocol for frozen sectioning is also included. Within paraffin sectioning, two fixation protocols are described (Bouin's and paraformaldehyde); the choice of fixative will be directly related to the antibody specifications and may require another fixing method.

We describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory.

Imuno-histoquímica em parafina da epiderme do rato utilizando anticorpos fluorescentes

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, ...

Drosophila Larval NMJ Immunohistochemistry

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 31 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. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.

Drosophila Larval NMJ Immunohistochemistry

This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.

Drosophila larval MNJ Imunohistoquímica

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

Optical Recording of Electrical Activity in Guinea-pig Enteric Networks using Voltage-sensitive Dyes

The enteric nervous system (ENS) is a self-contained network with identified functions, capable of performing complex behaviors in isolation. Its neurons (10 to 25 &mu;m in diameter) are arranged in plexuses that are confined to distinct planes of the gut wall 1; the myenteric plexus can be found between the longitudinal and circular muscle layers, and the submucous plexus between the circular muscle layer and the mucosa. Since the effector systems for these plexuses (transporting epithelium, endocrine cells, immune elements, blood vessels and smooth muscle) are also contained within the gut wall, semi-intact preparations can be dissected that preserve individual components of different reflex pathways. The behavior of the effector systems is controlled by the submucous and myenteric plexuses acting in concert. Therefore, detailed knowledge of synaptic interactions within and between ganglia, and of communication between the plexuses, is essential for understanding normal gastrointestinal function. The ENS, as an intact nervous system, is a unique experimental model in which one can correlate molecular and cellular events with the electrical behavior of the neuronal network and its physiological outputs. Because of the quasi-two-dimensional organization of its plexuses, the ENS is particularly well suited for the study of neural networks using multiple site optical recording techniques that employ voltage-sensitive dyes 2,7,8,9. We will illustrate here the use of a relatively new naphthylstyryl-pyridinium dye (di-4-ANEPPDHQ) 3 that offers multiple advantages over its predecessors, including very low phototoxicity, slow rate of internalization, and remarkable chemical stability. When used in conjunction with a camera that permits sub-millisecond time resolution, this dye allows us to monitor the electrical activity of all the neurons in the field of view with a maximal spatial resolution of ~ 2.5 &mu;m at 100X magnification. At lower magnification (10X or 20X), the sacrifice of single-cell resolution is compensated by a gain in perspective, revealing the intricacies of the inter-ganglionic circuitry.

This protocol illustrates how voltage-sensitive dyes enable optical recording of electrical activity from intact neural networks such as the plexuses of the guinea-pig enteric nervous system, with an adjustable resolution that ranges from single-cells to multi-ganglionic circuitry.

A gravação óptica de atividade elétrica na Guiné-porco Networks Enteric usando Tensão sensível Corantes

The enteric nervous system (ENS) is a self-contained network with identified functions, capable of performing complex behaviors in isolation. Its neurons ...

Drosophila Pupal Abdomen Immunohistochemistry

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic morphologies 1-3. During pupation, which spans approximately 96 hours (at 25 &deg;C), proliferating populations of imaginal cells replace the larval epidermis to generate the adult abdominal segments. These imaginal cells, born during embryogenesis, exist as lateral pairs of histoblast nests in each abdominal segment of the larvae. Four pairs of histoblast nests give rise to the adult dorsal cuticle (anterior and posterior dorsal nests), the ventral cuticle (ventral nests) and the spiracles associated with each segment (spiracle nests) 4. Upon puparation, these diploid cells (distinguishable by size from the larger polyploid larval epidermal cells- LECs) begin a stereotypical process of proliferation, migration and replacement of the LECs. Various molecular and genetic tools can be employed to investigate the contributions of genetic pathways involved in morphogenesis of the adult abdomen. Ultimate adult phenotypes are typically analyzed following dissection of adult abdominal cuticles. However, investigation of the underlying molecular processes requires immunohistochemical analyses of the pupal epithelium, which present unique challenges. Temporally dynamic morphogenesis and the interactions of two distinct epithelial populations (larval and imaginal) generate a fragile tissue prone to excessive cell loss during dissection and subsequent processing. We have developed methods of dissection, fixation, mounting and imaging of the Drosophila pupal abdominem epithelium for immunohistochemical studies that generate consistent high quality samples suitable for confocal or standard fluorescent microscopy.

Drosophila Pupal Abdomen Immunohistochemistry

Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.

Drosophila Imunohistoquímica Abdomen Pupal

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic ...

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&#38;E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&#38;E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.

This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.

qPCR é um método sensível e rápido para detecção de DNA por citomegalovírus em formalina-fixo, tecido da biópsia de parafina-embedded

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for ...

Colorimetric Detection of Bacteria Using Litmus Test

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. Such methods need to be user-friendly and produce reliable results that can be easily interpreted by both specialists and non-professionals. The litmus test for pH is simple, quick, and effective as it reports the pH of a test sample via a simple color change. We have developed an approach to take advantage of the litmus test for bacterial detection. The method exploits a bacterium-specific RNA-cleaving DNAzyme to achieve two functions: recognizing a bacterium of interest and providing a mechanism to control the activity of urease. Through the use of magnetic beads immobilized with a DNAzyme-urease conjugate, the presence of bacteria in a test sample is relayed to the release of urease from beads to solution. The released urease is transferred to a test solution to hydrolyze urea into ammonia, resulting in an increase of pH that can be visualized using the classic litmus test.

We describe a protocol for colorimetric detection of E. coli using a modified litmus test that takes advantage of an RNA-cleaving DNAzyme, urease, and magnetic beads.

Detecção Colorimétrico de bactérias usando teste decisivo

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. ...

Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between defective mitophagy and various human diseases, including neurodegenerative diseases, cancer, and metabolic diseases. In addition, recent studies have shown that mitophagy is involved in normal cellular processes, such as differentiation and development. However, the precise role of and molecular mechanisms underlying mitophagy require further study. Therefore, it is critical to develop a robust and convenient method for measuring changes in mitophagy activity. Here, we describe a detailed protocol for quantitatively assessing mitophagy activity through flow cytometry using the mitochondria-targeted fluorescent protein Keima (mt-Keima). This flow cytometry assay can analyze mitophagy activity more rapidly and sensitively than conventional microscopy- or immunoblotting-based methods. This protocol can be applied to analyze mitophagy activity in various cell types.

Mitophagy, the selective degradation of mitochondria, has been implicated in mitochondrial homeostasis and is deregulated in various human diseases. However, convenient experimental methods for measuring mitophagy activity are very limited. Here, we provide a sensitive assay for measuring mitophagy activity using flow cytometry.