O estudo foi financiado em parte pelo Programa de Pesquisa Intramural dos Institutos Nacionais de Saúde, Instituto Nacional do Câncer, Centro de Pesquisa do Câncer.

O protocolo descrito foi utilizado em conformidade com as normas do NIH sobre a utilização de amostras de tecidos humanos. 1. Preparação de Tecidos <ol><li> Antes do início, obter amostras de tecidos humanos de acordo com a Institutional Review Board (IRB) protocolos. </li><li> Escolha o tipo de bloco de tecido / célula e correspondente método de processamento [fixadas em formalina embebido em parafina (FFPE), congelados ou fixo-etanol (EFPE) incluído em parafina]. Fixação formalina fornece histologia ideal, com fixação de etanol e flash congelado. No entanto, métodos de transformação e de fixação do tecido podem afectar o ADN, ARN, e a quantidade de proteína e de qualidade para análise molecular e a jusante devem ser considerados. </li><li> Corte as seções do bloco de tecido / célula para o tipo de slide (vidro, vidro da membrana, ou membrana estrutura de metal) de escolha. Análise SIVQ funciona igualmente bem em todos os três tipos de slides. Por favor, note que o método pseudo-lamela (descrito abaixo) com xilenos e Ethanol não pode ser realizado nas lâminas de membrana de armação de metal uma vez que a lâmina tem de ser invertida no palco. </li><li> Selecione uma mancha tecido química ou com base em IHC para identificar as células de interesse do fundo. Note-se que os métodos de coloração pode também afectar o ADN, ARN, e qualidade e quantidade de tecido de proteína. Teste o tecido após coloração para avaliar a qualidade da linha de base de biomoléculas, antes de prosseguir com o protocolo. SIVQ-LCM foi realizada em lâminas de tecido / citologia coradas com: imuno-histoquímica (IHQ) com DAB 15, imunofluorescência, vermelho rápido, de novo vermelho, azul de toluidina e hematoxilina e eosina (H &amp; E) (dados não publicados). </li></ol> 2. Specimen Imagem <ol><li> Carga desliza no palco motorizado do instrumento microdissection e lançar o software relacionado. Marque as caixas de seleção para designar as posições das lâminas carregadas e garantir que os arquivos de imagens captadas devem ser salvos no formato jpeg. </li><li> Otimizar o imqualidade idade, ajustando o brilho eo foco da imagem na tela, usando o anel de foco manual ou através do software do instrumento de dissecação. <ol><li> Usando a caixa de ferramentas Imagem no software do instrumento de dissecação, definir o ganho de brilho da lâmpada e câmera de forma adequada. Exemplo de valores de brilho são = 60 = 220 e ganho, com o difusor. </li><li> Concentre-se manualmente ou com o recurso de foco automático no software. </li></ol></li><li> Capturar uma imagem panorâmica miniatura do slide para fornecer um roteiro para o processo de dissecação. <ol><li> Para melhor qualidade de imagem de um slide de uncoverslipped, utilizar o difusor abaixo do condensador sobre o instrumento, ou, adicionar uma pequena quantidade (~ 30 mL) de etanol ou xilenos para melhorar o índice refratário (pseudo-lamela). Ao utilizar xilenos, estar ciente de que eles são deve ser utilizado medidas de segurança adequadas e tóxicas, incluindo o uso de um exaustor e jaleco de proteção, óculos de proteção dos olhos, e luvas. </li><li> NÃO coloque a tampa da LCM no slide até a solução de etanol ou xilenos foi completamente limpo ou o polímero na tampa será distorcida. </li></ol></li><li> Navegue o slide e capturar imagens das áreas a serem dissecados em 10X, 20X ou 40X de ampliação. Se necessário, melhorar a imagem com software como AutoCorreção (no Microsoft Office Picture Manager), como descrito anteriormente 22. As imagens devem ser capturadas em formato jpeg, que lhes permitam ser re-importado para o software de seleção automática. </li></ol> 3. Algoritmo Análise da Imagem <ol><li> Transferência de imagens capturadas a partir do instrumento microdissection para a pasta SIVQ. Instalar e abrir os pacotes de software ArcturusXT, AutoScan e SIVQ no computador conectado ao instrumento de dissecação. Para ter acesso ao software SIVQ, por favor, entre em contato com o Dr. Ulysses Balis (ulysses@med.umich.edu). </li><li> Abrir SIVQ e carregar a imagem capturada (jpeg) de interesse. &lt;/ Li&gt; <li> Navegar para a área de interesse e / ou ajustar o tamanho das janelas de visor de visualização (5 e 6). No software SIVQ, Viewport 5 mostra a imagem de pré-processamento e Viewport 6 mostra a imagem pós-processamento 16. </li><li> Escolher o tamanho do vector de anel e o número de anéis de ser utilizados. </li><li> Selecione o recurso de imagem predicado a ser capturada pelo botão direito sobre ele na Viewport 6. </li><li> Clique em &quot;Scan&quot; para analisar a imagem. </li><li> Ajustar a probabilidade estatística da imagem correspondente, utilizando as duas barras deslizantes. A barra superior ajusta a especificidade global do vetor, e é utilizado para excluir a área a partir da pesquisa inicial (com sensibilidade, tal como definido pela variável selecionado &quot;Dados&quot;) que podem representar excessiva área incluída. Por outro lado, a barra inferior é utilizado para aumentar a sensibilidade, depois de uma análise é executada, com a intenção de aumentar a área que é classificada como sendo um jogo. Ambos os controles deslizantes utilize a variável &quot;Dados&quot;, como o limiar de sensibilidade ao início do estudo. </li><li> Para salvar a imagem, clique em &quot;salvar como jpeg&quot; (imagem agora está salvo na pasta c :/ vq_test / fotos). </li><li> Analisar a imagem com o algoritmo. A saída do algoritmo de análise deve resultar numa imagem anotada para que possa ser utilizado em SIVQ-LCM. No momento, a versão atual do motor central SIVQ está em testes beta versão com a expectativa de que a versão de produção total (disponível Q1 2014) irá incorporar um kit de desenvolvimento de software completo (SDK) e uma interface de programação de aplicativo (API) para a integração simplificada de filtros espaciais gerados por usuários e as etapas de processamento de dados de fluxo de trabalho a jusante com o motor correspondente anel central. Este SDK será distribuído com um conjunto completo de documentação. <ol><li> Verifique se o heatmap SIVQ é alterado para uma cor vermelha uniforme. </li></ol></li><li> Exporte a imagem. É essencial para a re-inserir as coordenadas de posição do post-análise de imagem JPEG usando um editor de HEX para colar no cabeçalho do arquivo a partir da imagem instrumento dissecção originais. Os dados apropriados podem ser encontradas entre o &quot;Início da Imagem&quot; marcador (0xFF, 0xD8) eo primeiro &quot;Definir Quantização Table&quot; marcador (0xFF, 0xDB). </li></ol> 4. Microdissecção <ol><li> Coloque a tampa LCM no centro da região de interesse, onde as imagens foram capturadas para análise SIVQ. </li><li> Calibrar e Controle de Qualidade (CQ) os lasers UV / IR e otimizar os parâmetros, incluindo a energia, a duração, locais de laser, e velocidade de corte UV (como recomendado pelo fabricante). Execute estas calibrações antes de voltar a importar a imagem analisada. </li><li> Abrir AutoScanXT (software seleção automática) e importar a imagem analisada a partir de c :/ vq_test pasta / pics. </li><li> Treinar o software de selecção automática para reconhecer a anotação SIVQ e criar o mapa de dissecação. <ol><li> Para criar um training arquivo, selecione quatro regiões de interesse (marcadas por &quot;círculos azuis&quot;) sobre a &quot;tinta vermelha&quot; da imagem analisada SIVQ. </li><li> Selecione as áreas de fundo (marcadas por &quot;quadrados vermelhos&quot;) que não estão a ser dissecado. </li><li> Clique no botão &quot;Analisar&quot; para gerar o arquivo de treinamento, que pode ser guardado para usos posteriores. </li></ol></li><li> Realizar microdissecção usando o apropriado infravermelho (IR) e / ou ultravioleta (UV) lasers. <ol><li> Copie as áreas selecionadas para a imagem &quot;ao vivo&quot;. </li><li> Na caixa de ferramentas &quot;microdissect&quot;, selecione a captura IR apropriado ou botões de corte UV. </li></ol></li><li> Após a dissecção é completa, mova a tampa de LCM para a estação de QC e capturar uma imagem dos tecidos / células dissecadas. Outra abordagem é o de colocar a tampa sobre uma área vazia do slide antes de movê-lo para a estação de QC, o que permite ao usuário tirar fotos em várias ampliações. </li><li>Capturar uma imagem da área de tecido de interesse após microdissecção para avaliar ainda mais levantando a eficiência. </li><li> Se as células desejadas foram dissecados com êxito, clique no botão &quot;estágio atual&quot; no software ArcturusXT e retire a tampa LCM dar início ao processo de extração molecular para análise de downstream. </li></ol>

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Michael R. Emmert-Buck é um inventor de patentes NIH-realizadas cobrindo captura de microdissecção a laser e recebe pagamentos baseados em direitos autorais por meio do Programa de Transferência de Tecnologia NIH.

Nós apresentamos um protocolo para a aplicação de SIVQ-LCM para microdissect células epiteliais imunoistoquímica de FFPE tecido mamário humano. O uso de um algoritmo de análise de imagem, tal como SIVQ, reduz a quantidade de mão-de tempo necessário para o processo de microdissecção. Este é um avanço potencialmente importante para o campo de tempo e uma vez que o esforço do operador é tipicamente o passo limitante da velocidade para a dissecção precisa das células de interesse. No presente protocolo, es...

Originalmente desenvolvido em meados dos anos 1990, a captura a laser microdissecção (LCM), permite ao usuário capturar com precisão células específicas ou regiões celulares de uma secção de tecido histológico via visualização microscópica 1, 2. Muitos estudos comparando a análise molecular de LCM contra arranhões tecido ilustrar o valor do método 3-12. Além disso, há três publicações de protocolo de vídeo sobre a tecnologia que está disponível para visualização 13, 14. No entanto, apesar de seu valor comprovado, LCM pode ser tedioso e trabalhoso quando o alvo de interesse é uma população de células dispersas em uma seção de tecido heterogêneo, ou quando um grande número de células são necessárias para aplicações a jusante específicas, como a proteômica. A carga colocada sobre o operador humano nos levou a desenvolver uma abordagem dissecação semi-automatizado para LCM pela combinação de um algoritmo de análise de imagem poderosa para orientar o processo LCM 15. &lt;p class = &quot;jove_content&quot;&gt; Em colaboração com a Universidade de Michigan, nosso laboratório no NIH estendeu o previamente desenvolvido e relatado espacialmente invariante quantização vetorial (SIVQ) algoritmo de forma a permitir que ele semi-automatizar o processo de seleção de tecido intrínseco à guiados microdissection, tornando assim disponível uma ferramenta com o patologista ou cientista vida em mente. Espacialmente invariante quantização vetorial (SIVQ) é um algoritmo que permite que o usuário simplesmente &quot;clicar&quot; sobre uma característica histológica de interesse para criar um vetor de toque (recurso de imagem predicado) que pode ser usado para procurar toda a imagem histológica, o ajuste do limiar estatístico conforme necessário 16-21. O mapa de calor resultante exibe a qualidade de jogos para o recurso inicial imagem predicado e é posteriormente convertido em uma única cor (vermelho) mapa anotação que pode ser importado para o instrumento LCM. O software de seleção automatizada, AutoScanXT, é então usada para desenhar um mapa com basena anotação do SIVQ guiar a captura das células alvo da amostra de tecido. O protocolo detalhado abaixo descreve a implementação de SIVQ no fluxo de trabalho microdissection.

<table border="0" cellpadding="0" cellspacing="0" width="586">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Positive Charged Glass Slides</td>
			<td style="width:84px;">Thermo Scientific</td>
			<td style="width:119px;">4951Plus-001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Xylenes, ACS reagent, &#8805;98.5% xylenes + ethylbenzene basis&#160;</td>
			<td style="width:84px;">Sigma Aldrich</td>
			<td style="width:119px;">247642</td>
			<td style="width:167px;">CAUTION: PLEASE USE PROPER SAFETY PROCEDURES.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Ethyl Alcohol, U.S.P. 200 Proof, Anhydrous</td>
			<td style="width:84px;">The Warner-Graham Company</td>
			<td style="width:119px;">6.505E+12</td>
			<td style="width:167px;">CAUTION: PLEASE USE PROPER SAFETY PROCEDURES.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Arcturus CapSure Macro LCM Caps</td>
			<td style="width:84px;">Life Technologies</td>
			<td style="width:119px;">LCM0211</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">ArcturusXT Laser Microdissection Instrument</td>
			<td style="width:84px;">Life Technologies</td>
			<td style="width:119px;">ARCTURUSXT</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:216px;">AutoScanXT Software</td>
			<td style="width:84px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">An optional image analysis program for the ArcturusXT Laser Microdissection Device. This is software is required for SIVQ-LCM.</td>
		</tr>
		<tr height="169">
			<td height="169" style="height:169px;width:216px;">Spatially Invariant Vector Quantization (SIVQ)</td>
			<td style="width:84px;">University of Michigan</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">This tool suite is publicly available for academic collaborations. For access to the SIVQ algorithm, please contact Dr. Ulysses Balis [Ulysses@med.umich.edu]</td>
		</tr>
	</tbody>
</table>

sivq-lcm protocol for the arcturusxt instrument

SIVQ-LCM é uma nova metodologia que automatiza e simplifica o, o processo de dissecação de laser mais tradicional dependente do usuário. O objetivo é criar uma, rapidamente personalizável tecnologia laser avançada plataforma de dissecção. Neste relatório, nós descrevemos a integração do software de análise de imagem e espacialmente invariantes Quantização Vetorial (SIVQ) sobre o instrumento ArcturusXT. O sistema contém um ArcturusXT infravermelho (IV) e ultravioleta (UV) de laser, permitindo celular específico ou grandes dissecações da área. O objetivo principal é melhorar a velocidade, precisão e reprodutibilidade da dissecção de laser para aumentar a produtividade de amostras. Esta nova abordagem facilita microdissection de animais e tecidos humanos em investigação e fluxos de trabalho clínicos.

A secção de tecido FFPE materno foi histoquímica para citoqueratina AE1/AE3 usando um protocolo padrão IHC 23. Após a coloração, o tecido foi colocado deslizante na fase ArcturusXT e o protocolo SIVQ-LCM foi iniciado como descrito acima. Uma vez que o tecido não pode ser lamínulas por microdissecção, as células coradas + IHC pode ser difícil de discernir visualmente (Figura 1A). Assim, para proporcionar um melhor índice correspondente e uma melhoria da imagem, xilenos foram adic...

Watch this Scientific Journal Video about SIVQ-LCM Protocol for the ArcturusXT Instrument at JoVE.com

SIVQ-LCM Protocol for the ArcturusXT Instrument

SIVQ-LCM é uma abordagem inovadora que utiliza um algoritmo de computador, espacialmente invariantes Quantização Vetorial (SIVQ), para conduzir o processo a laser microdissection captura (LCM). O fluxo de trabalho SIVQ-LCM melhora a velocidade e precisão da microdissecção, com aplicações tanto na pesquisa e na clínica.

Protocolo SIVQ-LCM para o Instrumento ArcturusXT

SIVQ-LCM is a new methodology that automates and streamlines the more traditional, user-dependent laser dissection process.&#160;It aims to create an ...

sivq-lcm-protocol-for-the-arcturusxt-instrument

Research

JoVE Journal

Biology

8.6K visualizações.  National Institutes of Health.  SIVQ-LCM é uma abordagem inovadora que utiliza um algoritmo de computador, espacialmente invariantes Quantização Vetorial (SIVQ), para conduzir o processo a laser microdissection captura (LCM). O fluxo de trabalho SIVQ-LCM melhora a velocidade e precisão da microdissecção, com aplicações tanto na pesquisa e na clínica. 

Vídeo: SIVQ-LCM Protocol for the ArcturusXT Instrument

Protocol for Mosquito Rearing (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night cycle. For this procedure, you'll need mosquito cages, 10% sterile sucrose solution, paper towels, beaker, whatman filter paper, glass feeders, human blood and serum, water bath, parafilm, distilled water, clean plastic trays, mosquito food (described below), mosquito net to cover the trays, vacuum, and a collection chamber to collect adults.

Protocol for Mosquito Rearing A. gambiae

This video illustrates the general techniques used to rear Anopheles gambiae in the laboratory. The methods for caring for laboratory mosquitoes are demonstrated through all stages of the organism's life cycle from larvae to pupae to blood-feeding adults.

Protocolo para a Criação Mosquito (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night ...

Biologia

Neutrophil Isolation Protocol

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory immune response. Due to their key role in inflammation, neutrophil functions such as locomotion, cytokine production, phagocytosis, and tumor cell combat are extensively studied. To characterize the specific functions of neutrophils, a clean, fast, and reliable method of separating them from other blood cells is desirable for in vitro studies, especially since neutrophils are short-lived and should be used within 2-4 hours of collection. Here, we demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media that is a mixture of sodium metrizoate and Dextran 500. The procedure consists of layering whole blood over the density gradient medium, centrifugation, separation of neutrophil layer, and lysis of residual erythrocytes. Cells are then washed, counted, and resuspended in buffer to desired concentration. When performed correctly, this method has been shown to yield samples of >95% neutrophils with >95% viability.

Neutrophils are among the first cells to arrive on the site of inflammatory immune response, and their functions and mechanisms have been studied extensively in vitro. We demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media.

Isolamento de neutrófilos Protocolo

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory ...

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

Elaboração de Drosophila Os embriões para Live-Imaging Usando o protocolo gota suspensa

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

A Protocol for the Production of KLRG1 Tetramer

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. KLRG1 exists both as a monomer and as a disulfide-linked homodimer. This well-conserved receptor is found on the most mature and recently activated NK cells as well as on a subset of effector/memory T cells.
Using KLRG1 tetramer as well as other methods, E-, N-, and R-cadherins were identified as KLRG1 ligands. These Ca2+-dependent cell-cell adhesion molecules comprises of an extracellular domain containing five cadherin repeats responsible for cell-cell interactions, a transmembrane domain and a cytoplasmic domain that is linked to the actin cytoskeleton. 
Generation of the KLRG1 tetramer was essential to the identification of the KLRG1 ligands. KLRG1 tetramer is also a unique tool to elucidate the roles cadherin and KLRG1 play in regulating the immune response and tissue integrity.

This protocol describes the production of KLRG1 tetramer, which is a powerful tool for the analysis of KLRG1 ligands.

Um protocolo para a Produção de KLRG1 tetrâmero

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. ...

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle 1. In larval stages of insects belonging to the orders of Lepidoptera (moths and butterflies) and Diptera (true flies), hemocytes are formed from the lymph gland (a specialized hematopoietic organ) or embryonic cells and can be carried through to the adult stage. Embryonic hemocytes are involved in cell migration during development and chemotaxis regulation during inflammation. They also take part in cell apoptosis and are essential for embryogenesis 2. Hemocytes mediate the cellular arm of the insect innate immune response that includes several functions, such as cell spreading, cell aggregation, formation of nodules, phagocytosis and encapsulation of foreign invaders 3. They are also responsible for orchestrating specific insect humoral defenses during infection, such as the production of antimicrobial peptides and other effector molecules 4, 5. Hemocyte morphology and function have mainly been studied in genetic or physiological insect models, including the fruit fly, Drosophila melanogaster 6, 7, the mosquitoes Aedes aegypti and Anopheles gambiae 8, 9 and the tobacco hornworm, Manduca sexta 10, 11. However, little information currently exists about the diversity, classification, morphology and function of hemocytes in non-model insect species, especially those collected from the wild 12.
Here we describe a simple and efficient protocol for extracting hemocytes from wild caterpillars. We use penultimate instar Lithacodes fasciola (yellow-shouldered slug moth) (Figure 1) and Euclea delphinii (spiny oak slug) caterpillars (Lepidoptera: Limacodidae) and show that sufficient volumes of hemolymph (insect blood) can be isolated and hemocyte numbers counted from individual larvae. This method can be used to efficiently study hemocyte types in these species as well as in other related lepidopteran caterpillars harvested from the field, or it can be readily combined with immunological assays designed to investigate hemocyte function following infection with microbial or parasitic organisms 13.

Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.

Um protocolo simples para extrair Hemócitos de Lagartas selvagens

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle ...

Clock Scan Protocol for Image Analysis: ImageJ Plugins

The clock scan protocol for image analysis is an efficient tool to quantify the average pixel intensity within, at the border, and outside (background) a closed or segmented convex-shaped region of interest, leading to the generation of an averaged integral radial pixel-intensity profile. This protocol was originally developed in 2006, as a visual basic 6 script, but as such, it had limited distribution. To address this problem and to join similar recent efforts by others, we converted the original clock scan protocol code into two Java-based plugins compatible with NIH-sponsored and freely available image analysis programs like ImageJ or Fiji ImageJ. Furthermore, these plugins have several new functions, further expanding the range of capabilities of the original protocol, such as analysis of multiple regions of interest and image stacks. The latter feature of the program is especially useful in applications in which it is important to determine changes related to time and location. Thus, the clock scan analysis of stacks of biological images may potentially be applied to spreading of Na+ or Ca++ within a single cell, as well as to the analysis of spreading activity (e.g., Ca++ waves) in populations of synaptically-connected or gap junction-coupled cells. Here, we describe these new clock scan plugins and show some examples of their applications in image analysis.

This paper describes two novel ImageJ plugins for &#39;Clock Scan&#39; image analysis. These plugins expand the functionality of the original visual basic 6 program and, most importantly, make the program available to a large research community by bundling it with the ImageJ free image analysis software package.

Protocolo de varredura do relógio para análise de imagem: plugins ImageJ

The clock scan protocol for image analysis is an efficient tool to quantify the average pixel intensity within, at the border, and outside (background) a ...

A Protocol for Laboratory Housing of Turquoise Killifish (Nothobranchius furzeri)

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of reference fish model systems, such as zebrafish and medaka. In recent years, an emerging fish &#8211; the turquoise killifish (Nothobranchius furzeri) &#8211; has been adopted by a growing number of research groups in the fields of biology of aging and ecology. With a captive life span of 4 - 8 months, this species is the shortest-lived vertebrate raised in captivity and allows the scientific community to test &#8211; in a short time &#8211; experimental interventions that can lead to alterations of the aging rate and life expectancy. Given the unique biology of this species, characterized by embryonic diapause, explosive sexual maturation, marked morphological and behavioral sexual dimorphism - and their relatively short adult life span -&#160;ad hoc husbandry practices are in urgent demand. This protocol reports a set of key husbandry measures that allow optimal turquoise killifish laboratory care, enabling the scientific community to adopt this species as a powerful laboratory animal model.

A Protocol for Laboratory Housing of Turquoise Killifish Nothobranchius furzeri

Laboratory housing of turquoise killifish can be scaled up&#160;to house and efficiently raise thousands of individual fish in a centralized water filtration system, employing the same infrastructure used for standard zebrafish facilities. Here we detail a list of standardized procedures that allow efficient killifish maintenance.

Um protocolo para laboratório habitação de turquesa Killifish (Nothobranchius furzeri)

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of ...

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ecologically-important endpoints, though they are rarely considered in toxicological studies. D. melanogaster is emerging as a toxicology model for the behavioral, neurological, and genetic impacts of toxicants, to name a few. More importantly, species in the genus Drosophila can be utilized as a model system for an integrative framework approach to incorporate emergent properties and answer ecologically-relevant questions in&#160;toxicology research. The aim of this paper is to provide a protocol for exposing species in the genus Drosophila to pollutants to be used as a model system for a range of phenotypic outputs and ecologically-relevant questions. More specifically, this protocol can be used to 1) link multiple biological levels of organization and understand the impact of toxicants on both individual- and population-level fitness; 2) test the impact of toxicants at different stages of developmental exposure; 3) test multigenerational and evolutionary implications of pollutants; and 4) test multiple contaminants and stressors simultaneously.

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

In this paper, we provide a detailed protocol for exposing species in the genus Drosophila to pollutants with the goal of studying the impact of exposure on a range of phenotypic outputs at different developmental stages and for more than one generation.

Protocolo experimental para o uso de drosófila como um sistema modelo invertebrados para testes de toxicidade em laboratório

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ...

A Labor-saving and Repeatable Touch-force Signaling Mutant Screen Protocol for the Study of Thigmomorphogenesis of a Model Plant Arabidopsis thaliana

Plants responding to both intracellular and extracellular mechanical stimulations (or force signals) and develop special morphological changes, a called thigmomorphogenesis. In past decades, several signaling components have been identified and reported for being involved in the mechanotransduction (e.g., calcium ion binding proteins and jasmonic acid biosynthesis enzymes). However, the relatively slow pace of research in the study of force signaling or thigmomorphogenesis is largely attributed to two reasons: the requirement for laborious human hand-manipulated touch induction of thigmomorphogenesis and the force strength errors associated with people&#8217;s hand-touch. To enhance the efficiency of external force loading on a plant organism, an automatic touch-force loading machine was built. This robotic arm-driven hair brush touches provide a labor-saving and easily repeatable touch-force simulation, unlimited rounds of touch repetition and adjustable touch strength. This hair touch-force loading machine can be used for both large scale screening of touch-force signaling mutants and the phenomics study of plant thigmomorphogenesis. In addition, touch materials such as human hair, can be replaced with other natural materials like animal hair, silk threads and cotton fibers. The automated moving arms on the machine may be equipped with water sprinkling nozzles and air blowers to mimic the natural forces of rain drops and wind, respectively. By using this automatic hair touch-force loading machine in combination with the hand-performed cotton swab touching, we have investigated the touch response of two force signaling mutants, MAP KINASE KINASE 1 (MKK1) and MKK2 plants. The phenomes of the touch-force loaded wild type plants and two mutants were evaluated statistically. They have exhibited significant differences in touch response.

A Labor-saving and Repeatable Touch-force Signaling Mutant Screen Protocol for the Study of Thigmomorphogenesis of a Model Plant Arabidopsis thaliana

A gentle touch-force loading machine is built from human hair brushes, robotic arms and a controller. The hair brushes are driven by robotic arms installed on the machine and move periodically to apply touch-force on plants. The strength of machine-driven hair touches is comparable to that of manually applied touches.

Um trabalho-economia e repetível Touch-Force sinalização mutante Screen Protocol para o estudo de Thigmomorphogenesis de uma planta modelo Arabidopsis Arabidopsis thaliana

Plants responding to both intracellular and extracellular mechanical stimulations (or force signals) and develop special morphological changes, a called ...

A Simple Protocol for Mapping the Plant Root System Architecture Traits

Comprehensive knowledge of plant root system architecture (RSA) development is critical for improving nutrient use efficiency and increasing crop cultivar tolerance to environmental challenges. An experimental protocol is presented for setting up the hydroponic system, plantlet growth, RSA spreading, and imaging. The approach used a magenta box-based hydroponic system containing polypropylene mesh supported by polycarbonate wedges. Experimental settings are exemplified by assessing the RSA of the plantlets under varying nutrient (phosphate [Pi]) supply. The system was established to examine the RSA of Arabidopsis, but it is readily adaptable to study other plants like Medicago sativa (Alfalfa). Arabidopsis thaliana (Col-0) plantlets are used in this investigation as an example to understand the plant RSA. Seeds are surface sterilized by treating ethanol and diluted commercial bleach, and kept at 4 &#176;C for stratification. The seeds are germinated and grown on a liquid half-MS medium on a polypropylene mesh supported by polycarbonate wedges. The plantlets are grown under standard growth conditions for the desired number days, gently picked out from the mesh, and submersed in water-containing agar plates. Each root system of the plantlets is spread gently on the water-filled plate with the help of a round art brush. These Petri plates are photographed or scanned at high resolution to document the RSA traits. The root traits, such as primary root, lateral roots, and branching zone, are measured using the freely available ImageJ software. This study provides techniques for measuring plant root characteristics in controlled environmental settings. We discuss how to (1) grow the plantlets, and collect and spread root samples, (2) obtain pictures of spread RSA samples, (3) capture the images, and (4) use image analysis software to quantify root attributes. The advantage of the present method is the versatile, easy, and efficient measurement of the RSA traits.

We use simple laboratory tools to examine the root system architecture (RSA) of Arabidopsis and Medicago. The plantlets are grown hydroponically over mesh and spread using an art brush to reveal the RSA. Images are taken using scanning or a high-resolution camera, then analyzed with ImageJ to map traits.

Um Protocolo Simples para Mapear as Características da Arquitetura do Sistema Radicular da Planta

Comprehensive knowledge of plant root system architecture (RSA) development is critical for improving nutrient use efficiency and increasing crop cultivar ...