We thank Dr. Vicky Macias, Angeline Giangreco and Avani Vaishnav for assistance with optimizing this methodology over the years and Yang Zhang and Dr. Jian Ma at the University of Illinois at Urbana-Champaign for the RNA seq analysis. This work was supported by NIH/NCI R01 CA166588-01 (Nonn), American Cancer Society Research Scholar 124264-RSG-13-012-01-CNE (Nonn), NIH/NCI R03 CA172827-01 (Nonn), DOD-CDMPR PRCP Health Disparities Idea Award PC121923 (Nonn) and a Prevent Cancer Foundation grant (Zhou).

Todos os tecidos humanos utilizados para estas experiências foram adquiridas através de um protocolo aprovado Institutional Review Board e / ou isenção na Universidade de Illinois em Chicago. 1. Seção Fresco Congelado próstata para PEN-slide e para Cobrado placa de vidro <ol><li> No dia anterior ou poucas horas antes do corte da amostra, ferramentas limpas (isto é, escova, fórceps, coplins, lâminas, lâminas PEN-emoldurados (se já não estiver RNase livre), um marcador ETOH seguro e um lápis) e no interior de um RT criostato com uma solução de RNase-descontaminação usando um frasco de spray e LAB-toalhetes. <ol><li> Permita que a solução de descontaminação RNase-a descansar por 5 minutos antes de enxugar, enxaguar com DEPCH 2 0 para remover sobra solução e uma lavagem final com 70% EtOH (preparado com DEPCH 2 0) RNase-descontaminação. Nota: É essencial para manter um espaço de trabalho livre de RNase. Todos os coplins, ferramentas, meio de montagem congelados, lâminas e slides utilizados devem ser usados ​​apenas para RNase de trabalho livree nunca usado em um ambiente livre não-RNase padrão. Todos os instrumentos têm de ser tratada com uma solução de descontaminação de RNase antes de cada experiência. </li></ol></li><li> Cryostat legal a -24 ° C. </li><li> Coloque os seguintes instrumentos RNase gratuitas limpos dentro cryostat esfriar por pelo menos 30 min antes do corte: slide com etanol a 100%, as ferramentas, as pequenas cassetes de tecido congelado e chuck montagem cheio de coplin. </li><li> Recuperar a partir de tecido da próstata congelado cryostorage e lugar em um balde espuma seca-cheia de gelo para o transporte. Colocar em criostato de tecido equilibrar à criostato temperatura durante pelo menos 30 min. Nota: tecidos da próstata humanos para a captura de laser de micro-dissecção pode ser fresco congelado ou formalina-fixados em parafina (FFPE). As limitações de tempo e preparação da lâmina são ligeiramente diferentes entre fontes de tecido. Aqui nós descrevemos os passos para cortes congelados. </li><li> Trabalhar com um tecido de cada vez, e esguichar meio de montagem congelado no bottom da cassete e rapidamente montar o tecido prostático congelado para o meio de montagem congelado utilizando a pinça refrigerados. </li><li> Coloque a gaveta com o tecido em resfriador em criostato durante 5 min para definir meio de montagem congelado. </li><li> Tutuca uma pequena quantidade de meio de montagem congelada para o chuck e pressione cuidadosamente tecido montado lado de baixo para cima do meio de montagem congelada. Deixe definido no criostato durante 5 min para permitir que o meio de montagem congelados para solidificar completamente. </li><li> Coloque chuck no suporte e aperte. </li><li> Trave a manivela na posição de bloqueio e coloque uma lâmina nova para o criostato. Para terminais moleculares a jusante que incluem um passo de amplificação (ou seja, qPCR), é aconselhável a utilização de uma lâmina nova para cada paciente para evitar a contaminação (ou mover a lâmina através de utilização da área da lâmina para a amostra seguinte). </li><li> Destravar a alça e cuidadosamente avançar o tecido à mão ou com um pedal para uniformizar e expor o tecido com 50 μsecções M até a exposição do tecido desejado seja alcançado. </li><li> Ajuste cryostat a 5 um, cortar uma ou duas seções e colocá-los em cobrados lâmina de vidro para hematoxilina e eosina (H &amp; E) coloração (ver Passo 2). </li><li> Colocar imediatamente a deslizar para o etanol frio a 100% durante 2 min. </li><li> Remover a lâmina de vidro carregada a partir de etanol e deixar secar à RT, antes de prosseguir para a coloração com H &amp; E (Passo 2). </li><li> Coloque RT PEN-quadro de slide em um torcedor do quadro. </li><li> Ajuste cryostat 10 seções um e lugar para os slides quadro RT PEN. Um a quatro secções pode ser colocado em cada corrediça, dependendo do tamanho do tecido. </li><li> Mergulhar imediatamente a deslizar para o etanol frio a 100% durante 2 min. </li><li> Retirar as lâminas PEN-quadro a partir de etanol e armazenar o slide em uma caixa de slide em uma caixa de secador em um freezer -80 ° C até que esteja pronto para LCM. Três dias é o tempo máximo de armazenamento para recuperação de RNA ideal. </li><li> Prossiga com a Etapa 2 para apenas o vidro cobrado deslizoue. Prossiga com a Etapa 3 para PEN-quadro de slide. </li></ol> 2. Hematoxilina e Eosina-Y (H &amp; E) da mancha por histológica Mark-up Nota: Este caso para o tecido sobre a lâmina de vidro carregada e não o slide PEN-quadro para LCM. <ol><li> Hidratar as secções sobre lâminas de vidro carregada mergulhando a lâmina por meio de etanol graduada como segue: mergulha o slide duas vezes em etanol a 100% durante 3 min, duas vezes em etanol a 95% durante 3 min, uma vez em etanol a 70% durante 3 min e dois vezes em H2O destilada durante 3 min. <ol><li> Preparar a solução de álcool de ácido: 99 ml de etanol a 70% + 1 ml de 1% de HCl e solução de bicarbonato de sódio a 0,1%: 0,1 g de bicarbonato de sódio em 100 ml de H2O destilada </li></ol></li><li> Mergulhe as lâminas em hematoxilina Gill II (0,4%) por 5 min. </li><li> Lavar o excesso de corante em constante funcionamento torneira H2O por 3 min. </li><li> Mergulhe o slide 10 vezes em álcool ácido </li><li> Lave o slide no corrente da torneira H2O por 3 min. </li><li> Mergulhe o slidenuma solução de bicarbonato de sódio a 0,1% durante 30-60 seg para transformar a cor púrpura para azul. </li><li> Lave o slide no corrente da torneira H2O por 3 min. </li><li> Lavar as lâminas em etanol a 95%, com 10 mergulhos. </li><li> Mergulhe o slide no Eosina-Y 2-3 vezes 15 seg total. </li><li> Desidratar o slide através de etanol classificados da seguinte forma: mergulhe a lâmina duas vezes em etanol a 95%, duas vezes em etanol a 100%, e duas vezes em xileno (certifique-se o tecido parece claro o que indica desidratação completa). </li><li> Montar o slide com não aquoso difícil montagem médio e tampa de deslizamento permanente. </li><li> Deixe que a corrediça seco durante 30 min. </li><li> Digitalização de slides com qualquer scanner de slides todo e imprimir uma imagem grande, de alta resolução para 8 &quot;x 11&quot; papel. </li><li> Esboço áreas de interesse (normalmente feito por um patologista certificado bordo) com um marcador. Essa marcação é o guia para o procedimento LCM. </li></ol> 3. Azul de Toluidina coloração das lâminas PEN-quadro Nota: Thestá é feito o mesmo dia como o LCM. Use DEPCH 2 O de água para todas as soluções. Conclua todas as etapas em área livre de RNase. Todos os coplins devem ser limpas com uma solução de RNase-descontaminação. <ol><li> O dia de LCM remover as lâminas PEN-quadro de -80 ° C e hidrato de congelador por lâminas inclinadas em declive em EtOH a 90% durante 2 minutos, em seguida, 75% de EtOH durante 2 min. <ol><li> Prepare 0,5% azul de toluidina por dissolução em água biologia molecular grau em seguida, filtrar através de um filtro de esterilizar e armazenar 0,2 m à temperatura ambiente. Esta solução pode ser reutilizada durante até 6 meses. Nota: É altamente aconselhável para preparar a solução antes que a filtração pode levar algumas horas. </li></ol></li><li> Manchar o tecido da próstata mergulhando suavemente slides em 0,5% azul de toluidina para 5-30 seg. Destain o tecido desliza por lavagem 2 vezes em DEPCH 2 O durante 15 segundos seguido por 75% de EtOH durante 30 segundos a 3 minutos. Nota: mancha e destain tempos podem ser ajustados para assegurar uma boa coloração. Próstata tende a sobre a mancha. </li><li&gt; Transferir as lâminas PEN-quadro para recipiente seco RNAase-livre no gelo. </li></ol> Captura Laser-4. Micro-dissecção (LCM) <ol><li> Imediatamente antes de LCM, a corrediça seco sobre uma lâmina quente a 42 ° C durante 15 min. Dry apenas o slide que será processado e armazenar o resto em gelo até estar pronto para eles. </li><li> Ligue o computador e LCM, por esta ordem, o poder do microscópio, a chave de laser, o interruptor do laser e, em seguida, o computador. O lançamento do software Laser Microdissection. </li><li> Use o software para controlar o microscópio para carregar os slides e tubos de coleta. Clique em &quot;suporte de amostras de descarga&quot;, carregue o slide com as seções no suporte de slides do microscópio com o tecido voltado para baixo e insira o suporte de slides na ranhura apropriada no microscópio, em seguida, clique em &quot;continuar&quot;. <ol><li> Clique em &quot;tubos de coleta de descarregamento&quot;, rótulo e carregar 0,5 ml tubos no suporte do tubo e inseri-lo no slot apropriado no instrumento. Uma veztampas estão garantidos, adicionar 35 ul de tampão de lise para as tampas de coleta e tentar evitar bolhas. Em seguida, clique em &quot;OK&quot;. Nota: Se a evaporação é um problema, dilui-se o tampão da seguinte forma: 25 ul de tampão de lise com 10 ul depcH2O. </li></ol></li><li> No software, seleccionar a posição em que a corrediça tem sido colocada no suporte da lâmina. Selecione a tampa de cobrança para recolher a amostra LCM. </li><li> Visualize e focar o slide através do computador em 10X. Use o software para calibrar o laser, selecionando &quot;Laser&quot; e depois &quot;calibrar&quot;. Ajustar a potência, a abertura ea velocidade do laser, escolhendo por &quot;laser&quot;, em seguida, &quot;controlo&quot; para permitir que o feixe de laser para cortar a área seleccionada completa e eficiente. Repetir até que os ajustes cortes a laser completamente através do tecido. </li><li> Usando o patologista mark-up 8 &quot;x 11&quot; como um guia, use a ferramenta de &quot;chamar a forma&quot; de circunscrever as áreas desejadas de Epítetoslium na vista e evitar a coleta de qualquer estroma. Nota: Várias áreas podem ser selecionadas dentro de um ponto de vista, mas não se movem para outra vista sem primeiro corte clicando a ferramenta &quot;cortar forma&quot;. <ol><li> Se uma área não fica completamente cortada pelo laser de usar a ferramenta &quot;mover e cortar&quot; para passar por isso manualmente. Continue a mover o objetivo de novos pontos de vista e repita o corte a laser até slide é completamente dissecado ou a quantidade desejada de tecido foi coletada (tipicamente 100 glândulas benignas produzir 150-500 ng de RNA). (ver o exemplo na Figura 1A). </li></ol></li><li> Remova cuidadosamente os tubos de suporte à recolha e fechar as tampas, lembrando do tampão de lise e tecido nas calotas. Resumidamente, centrifugar durante 30 segundos para recolher o líquido e o tecido na parte inferior do tubo. Incubar as amostras a 42 ° C durante 30 min e armazenar a -80 ° C até estar pronto para o isolamento de ARN. </li></ol> 5. Isolamento de ARN total com um FilKit baseada em ter e Análise de Qualidade <ol><li> Descongelar os tubos em gelo e levar o volume da solução para 100 uL. </li><li> Pré-molhou a micro filtro de um kit de isolamento de ARN através da aplicação de 30 ul ofLysis Solução para o centro do filtro e deixe de molho whileperforming os dois passos seguintes (pelo menos, 5 min). </li><li> Adicionar 3 ml de solução de LCM Aditivo (fornecido no kit) ao lisado e misturar em vortex durante 5 segundos. Centrifugar durante 30 segundos para recolher o fluido na parte inferior do tubo. </li><li> Centrifugar o filtro pré-humedecido para ~ 30 segundos na velocidade superior para remover o líquido. </li><li> Adicionar 1,25 volumes (neste caso 129 ul) de etanol a 100% para a mistura de lisado, suavemente vórtice ou pipeta cima e para baixo. ** Este método irá recuperar as duas espécies de RNA grandes e pequenos. </li><li> Carregar a amostra sobre o micro-filtro pré-humedecido, e centrifuga-se a 10.000 xg durante 1 minuto para se ligarem a ARN ao filtro. Em seguida, lave o filtro micro com 180 ul de &quot;solução de lavagem 1, R21; centrifugar a 10000 xg durante 1 min. Nota: Deste ponto em diante todas as centrifugações deve ser feito a 13.000 xg, ou velocidade máxima. </li><li> Lavar duas vezes com filtrar 180 ul de &quot;soluções de lavagem 2 e 3&quot;, respectivamente, conforme indicado pelo protocolo do kit. Centrifugar durante 30 segundos, em seguida, descartar o fluxo através de, e girar o filtro durante 1 min para remover o líquido residual e para secar o filtro. </li><li> De transferência do filtro para um novo tubo de recolha. </li><li> Solução quente de eluição a -95 ° C (fornecido com o kit). </li><li> Aplicar 10 ul de solução de eluição pré-aquecido para o centro do filtro, fechar a tampa e armazenar durante 5 min à TA. Em seguida centrifugar durante 1 min para eluir o ARN, e repetir este passo de cada vez, para se obter ~ 20 ul de ARN. </li><li> Realizar o tratamento de DNase I durante 15 minutos a 37 ° C. </li><li> Quantificar o ARN com um espectrof otómetro de absorvência a 260 nm. A relação de densidade óptica no comprimento de onda de 260 nm e 280 nm é utilizado para avaliar a pureza deo RNA (ver Tabela 1) 15. </li></ol> 6. Análise da expressão gênica Nota: A expressão dos genes de espécies de ARN de comprimento (como mRNAs e lncRNAs) é mostrado no passo 6.1 e de espécies de RNA curtas (como microARNs) é mostrado no passo 6.2. Diferentes kits estão disponíveis para RT-qPCR e da quantidade de RNA necessário varia de acordo com kit (tão pouco quanto 10 ng). A análise de sequenciação de ARN é descrito em 6.3. <ol><li> Transcrever reversa (RT) do ARN para ADNc da mistura de 5 mL de 20 ng / ul de amostra de ARN com 5 uL da mistura principal RT (tampão RT, dNTP, iniciadores aleatórios, enzima RT, inibidor de RNase). Incubar a reacção durante 25 min a 25 ° C, em seguida, 120 min a 37 ° C e 5 min a 85 ° C. </li><li> Dilui-se a reacção de RT 1:10 com RNase isenta de água e usar 2,5 mL por 10 jil de reacção qPCR com iniciadores ou conjuntos iniciador / sonda para os genes de interesse. Nota: Para facilitar a análise de RNA parcialmente degradada É aconselhável a utilização de primers for amplicões mais curtos (menos do que 100 pb) 16. (Tabela 2B) <ol><li> Para análise de expressão microARN executar a reacção de RT através da mistura de 5 mL de 2-10 ng / ul de amostra de ARN com 5 uL da mistura principal RT. Nota: cada tecnologia de detecção com base em PCR microARN disponível comercialmente requer a utilização de iniciadores de RT e de propriedade específicos. </li></ol></li><li> Alternativamente ou em adição a RT-PCR, utilizar sequenciação geração seguinte (SEQ ARN-ARN ou de pequena-SEQ) para quantificar diferentes espécies de ARN. Tipicamente, usar 500 ng de ARN para este procedimento (Tabela 3) e levar a cabo o método tal como indicado pelo fabricante e detecção instrumento. </li></ol>

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The authors have nothing to disclose.

A expressão do gene de perfis a partir de amostras humanas pode ser um desafio, não só para a qualidade ou quantidade de tecido disponível, mas também para as várias entidades histológicas presentes numa determinada amostra de tecido. Isto é particularmente difícil na próstata em que os tecidos benignos são em grande parte tecidos estromais e áreas de cancro são desprovidos de estroma. LCM facilita a separação física do estroma prostático e ARN epitélio para uma assinatura mais preciso dos dois tipos d...

A próstata é um tecido heterogéneo composto por epitélio secretor dispostos em ácinos glandular rodeado por estroma fibromuscular composto principalmente de músculo liso 1. O compartimento epitelial é composto por cinco tipos de células diferentes, mas organizados: células basais, células secretoras, células neuroendócrinas, células amplificadoras de trânsito e células-tronco 2. No cancro da próstata (CaP), que surge a partir de células epiteliais luminais, o crescimento do adenocarcinoma provoca um declínio progressivo evidente do compartimento estromal 3. Por estas razões, as amostras de tecido terá diferenças distintas na proporção de estroma e tipo de célula epitelial com base na extensão de CaP. Estas diferenças podem levar a pressupostos enviesada dos dados de expressão de genes adquiridos a partir de tecidos inteiros sem ter em conta a microdissecação de o tipo de célula desejado. Portanto, para eliminar esta polarização é essencial para separar tipos de células antes da extracção de ARN eAnálise da expressão do gene. Macrodissection microdissecação ou pode ser usado para separar fisicamente as áreas epiteliais bem caracterizadas do estroma circundante 4 -6. Macrodissection é tipicamente feito com uma lâmina de barbear sob um microscópio de dissecação e funciona bem para a separação de grandes CaP nódulos de estroma, mas não é capaz de remover a partir de epitélio benigna estroma circundante (ver exemplo de histologia benigna da próstata na Figura 1). Microdissection com um laser (LCM) é significativamente mais trabalho intensivo do que macrodissection, mas pode dissecar com muita precisão epitélio benigno 4. Publicações recentes de nosso laboratório demonstraram que o RNA pode ser extraído com sucesso por LCM de qualquer um (FFPE) biópsias embebidos em parafina fixadas em formalina ou tecido congelado 4,7-9. Os principais desafios na extracção LCM-ARN são: 1) para dissecar com precisão as áreas desejadas do tecido, e 2) para preservar RNA integridade durante o processo de LCM 4,10 e isolamento. ARN isolado a partir de populações de células puras podem ser utilizados para análise da expressão do gene através de vários métodos, incluindo a transcrição reversa-PCR quantitativo (RT-qPCR) 7,8, 11 microarray, e profunda -sequencing 12-14. O objetivo deste protocolo é para isolar RNA total de LCM prostática epitélio de tecido congelado para análise de expressão de genes a jusante.

<table>
	<tbody>
		<tr>
			<td>RNase-AWAY&#160;</td>
			<td>MBP</td>
			<td>7005-11</td>
			<td></td>
		</tr>
		<tr>
			<td>PEN-membrane 4,0 mm slides&#160;</td>
			<td>Leica</td>
			<td>11600289</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides, Superfrost Plus</td>
			<td>FisherBrand</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 proof</td>
			<td>Decon labs.</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>DEPC (diethyl pyrocarbonate)</td>
			<td>Sigma</td>
			<td>D-5758</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM3050</td>
			<td></td>
		</tr>
		<tr>
			<td>Coplins (Staining jar)</td>
			<td>IHCWORLD</td>
			<td>M900-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Coplins (Staining rack)</td>
			<td>IHCWORLD</td>
			<td>M905-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Aperio ScanScope</td>
			<td>Aperio(Leica)</td>
			<td>ScanScope&#174; CS</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluidine Blue</td>
			<td>Fluka</td>
			<td>89640-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Microdissection System</td>
			<td>Leica</td>
			<td>LMD7000</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 mL Thin-walled Tubes for LCM</td>
			<td>Thermo Scientific</td>
			<td>AB-0350</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAqueous&#174;-Micro Total RNA Isolation Kit</td>
			<td>Ambion</td>
			<td>AM1931</td>
			<td>Thermo Fisher Scientific Brand</td>
		</tr>
		<tr>
			<td>NanoDrop</td>
			<td>Thermo Scientific</td>
			<td>ND-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit 2.0 Fluorometer</td>
			<td>Life Technologies</td>
			<td>Q32866</td>
			<td>Thermo Fisher Scientific Brand</td>
		</tr>
		<tr>
			<td>High-Capacity cDNA Reverse Transcription Kit</td>
			<td>Applied Biosystems</td>
			<td>4368814</td>
			<td>Thermo Fisher Scientific Brand</td>
		</tr>
		<tr>
			<td>Universal cDNA Synthesis Kit II, 8-64 rxns</td>
			<td>Exiqon</td>
			<td>203301</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan microRNA RT kit</td>
			<td>Applied Biosystems</td>
			<td>4366597</td>
			<td>Thermo Fisher Scientific Brand</td>
		</tr>
		<tr>
			<td>Hematoxylin stain&#160;</td>
			<td>Ricca Chemical Company</td>
			<td>3536-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin-Y</td>
			<td>Richard Allan Scientific&#160;</td>
			<td>7111</td>
			<td></td>
		</tr>
	</tbody>
</table>

laser-capture microdissection of human prostatic epithelium for rna analysis

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types. Healthy prostate is comprised of fibromuscular stroma surrounding discrete epithelial-lined secretory lumens and a very small population of immune and neuroendocrine cells. In contrast, areas of prostate cancer have increased dysplastic luminal epithelium with greatly reduced or absent stromal population. Given the profound difference between stromal and epithelial cell types, it is imperative to separate the cell types for any type of downstream molecular analysis. Despite this knowledge, the bulk of gene expression studies compare benign prostate to cancer without micro-dissection, leading to stromal bias in the benign samples. Laser-capture micro-dissection (LCM) is an effective method to physically separate different cell types from a specimen section. The goal of this protocol is to show that RNA can be successfully isolated from LCM-collected human prostatic epithelium and used for downstream gene expression studies such as RT-qPCR and RNAseq.

Num estudo anterior demonstrou que a utilização de LCM para recolher os tecidos epiteliais e do estroma para comparar perfis de expressão por RT-qPCR de ARNm e microARNs de tecidos da próstata e congelados FFPE a partir do mesmo paciente 4. LCM é demorado, especialmente se grande quantidade de RNA deve ser recolhida para análise de sequenciamento de próxima geração. Portanto, é crucial manter o espaço de trabalho e ferramentas de RNase livre. Recomenda-se para analisar os controlos de qualidade e c...

Watch this Scientific Journal Video about Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis at JoVE.com

Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

The goal of this protocol is to use laser-capture micro-dissection as an effective method to isolate pure populations of cell types from heterogeneous prostate tissues for downstream RNA analysis.

Captura de Laser Microdissection de Próstata Humano Epithelium para Análise de RNA

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types.  Healthy prostate is comprised of ...

laser-capture-microdissection-human-prostatic-epithelium-for-rna

Research

JoVE Journal

Medicine

13.3K visualizações.  University of Illinois at Chicago. The goal of this protocol is to use laser-capture micro-dissection as an effective method to isolate pure populations of cell types from heterogeneous prostate tissues for downstream RNA analysis.

Vídeo: Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

Dissection of Human Vitreous Body Elements for Proteomic Analysis

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions between vitreous substructures and the retina underlie several vitreoretinal diseases, including retinal tear and detachment, macular pucker, macular hole, age-related macular degeneration, vitreomacular traction, proliferative vitreoretinopathy, proliferative diabetic retinopathy, and inherited vitreoretinopathies. 1,2 The molecular composition of the vitreous substructures is not known. Since the vitreous body is transparent with limited surgical access, it has been difficult to study its substructures at the molecular level. We developed a method to separate and preserve these tissues for proteomic and biochemical analysis. The dissection technique in this experimental video shows how to isolate vitreous base, anterior hyaloid, vitreous core, and vitreous cortex from postmortem human eyes. One-dimensional SDS-PAGE analyses of each vitreous component showed that our dissection technique resulted in four unique protein profiles corresponding to each substructure of the human vitreous body. Identification of differentially compartmentalized proteins will reveal candidate molecules underlying various vitreoretinal diseases.

This video shows an effective technique for differentiating and dissecting the various semi-transparent structures of the human vitreous body in post mortem eyes.

Dissecção da Human Elements Corpo Vítreo para Análise Proteômica

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions ...

Medicina

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

Protocolo melhorado para microdissecção a laser de ilhotas pancreáticas humanas a partir de espécimes cirúrgicos

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-na&iuml;ve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis.
The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

Formação de Epithelium próstata humano utilizando Tissue Recombinação de Rodent Urogenital Mesênquima Sinus e células-tronco humanas

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia

New therapies for two common prostate diseases, prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend critically on experiments evaluating their hormonal regulation. Sex steroid hormones (notably androgens and estrogens) are important in PrCa and BPH; we probe their respective roles in inducing prostate growth and carcinogenesis in mice with experiments using compressed hormone pellets. Hormone and/or drug pellets are easily manufactured with a pellet press, and surgically implanted into the subcutaneous tissue of the male mouse host. We also describe a protocol for the evaluation of hormonal carcinogenesis by combining subcutaneous hormone pellet implantation with xenografting of prostate cell recombinants under the renal capsule of immunocompromised mice. Moreover, subcutaneous hormone pellet implantation, in combination with renal capsule xenografting of BPH tissue, is useful to better understand hormonal regulation of benign prostate growth, and to test new therapies targeting sex steroid hormone pathways.

We describe the manufacture of compressed hormone pellets, as well as subcutaneous surgical implantation into mice. This strategy can be combined with the growth of cell and tissue xenografts under the renal capsule of athymic nude mice to evaluate hormonal carcinogenesis and regulation of benign prostate growth.

Renal Capsule xeno e subcutânea Pellet Implantação de Avaliação de próstata Carcinogênese e hiperplasia benigna da próstata

New therapies for two common prostate diseases,  prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend  critically on experiments ...

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tg(fli:EGFP) Zebrafish Retinal Vasculature

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of diabetic microvascular complications one of the key research fields of the next decades. Specialized, targeted therapy and novel therapeutic drugs are needed to manage the increasing number of patients at risk of vision-loss. The zebrafish is an established animal model for developmental research questions with increasing relevance for modeling metabolic multifactorial disease processes. The advantages of the species allow for optimal visualization and high throughput drug screening approaches, combined with the strong ability to knock out genes of interest. Here, we describe a protocol which will allow easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis or vessel damage. This is achieved via dissection of the zebrafish retina and whole-mounting of the tissue. Visualization of the exposed vessels is then achieved via confocal microscopy of the green EGFP reporter expressed in the adult retinal vasculature. Correct handling of the tissue will lead to better outcomes and less internal vessel breakage to assure the visualization of the unaltered vascular structure. The method can be utilized in zebrafish models of retinal vasculopathy linked to changes in the vessel architecture as well as neoangiogenesis.

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tgfli:EGFP Zebrafish Retinal Vasculature

Here, we discuss a method protocol which will allow an easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis and structural changes.

Estudando a Diabetes através dos olhos de um peixe: Microdissection, visualização e análise do tg(fli:EGFP) adulto vasculatura da retina de Zebrafish

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of ...

Surgical Treatment for Benign Prostatic Hyperplasia: Holmium Laser Enucleation of the Prostate (HoLEP).

Benign prostatic hyperplasia (BPH) commonly occurs among elderly males. The condition causes symptoms in the lower urinary tract, thereby reducing the quality of life. BPH includes 2 major treatments: medication and surgery. Surgical treatment is often the most effective and final intervention. Holmium laser enucleation of the prostate (HoLEP), one of the most efficient surgical procedures for BPH, is conducted transurethrally. During surgery, the most important and most difficult part is to locate the surgical capsule of the prostate, which is consistent to the idea of anatomic surgery. Different skills may be needed in treating different parts of the prostate. HoLEP tends to be efficient and safe with good hemostatic properties, and able to treat bladder calculus, which may be the complications of BPH. The technique is particularly suitable for prostate of varying volumes and sizes. Indeed, HoLEP also presents certain disadvantages, such as a long learning curve and costly equipment. Regardless, this method may be the "new standard" and the excellent method for the surgical treatment of BPH.

Surgical Treatment for Benign Prostatic Hyperplasia: Holmium Laser Enucleation of the Prostate HoLEP.

Here we present a safe and efficient protocol, holmium laser enucleation of the prostate, to treat benign prostatic hyperplasia.

Tratamento cirúrgico para a hiperplasia prostática benigna: Holmium Laser Enucleação da próstata (HoLEP).

Benign prostatic hyperplasia (BPH) commonly occurs among elderly males. The condition causes symptoms in the lower urinary tract, thereby reducing the ...

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry (LCM-LC/MS)

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel therapeutics are urgently required. The ability of dosed TB drugs to reach and sterilize bacteria within poorly-vascularized necrotic regions (caseum) of pulmonary granulomas is crucial for successful therapeutic intervention. Effective therapeutic regimens must therefore contain drugs with favorable caseum penetration properties. Current LC/MS methods for quantifying drug levels in biological tissues have limited spatial resolution capabilities, making it difficult to accurately determine absolute drug concentrations within small tissue compartments such as those found within necrotic granulomas. Here we present a protocol combining laser capture microdissection (LCM) of pathologically-distinct tissue regions with LC/MS quantification. This technique provides absolute quantification of drugs within granuloma caseum, surrounding cellular lesion and uninvolved lung tissue and, therefore, accurately determines whether bactericidal concentrations are being achieved. In addition to tuberculosis research, the technique has many potential applications for spatially-resolved quantification of drugs in diseased tissues.

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry LCM-LC/MS

Here, we describe a protocol using laser capture microdissection coupled with LC/MS analysis to spatially-quantify drug distributions within pulmonary tuberculosis granulomas. The approach has broad applicability to quantifying drug concentrations within tissues at high spatial detail.

Spatial quantificação de drogas em lesões de tuberculose pulmonar por Laser capturar Microdissection líquido cromatografia de espectrometria de massa (LCM-LC/MS)

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel ...

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a promising therapeutic target in many disorders, such as autoimmune diseases, atherosclerosis, and cancer. RNAi-mediated gene silencing is a valuable approach of choice to probe and manipulate macrophage function; however, the transfection of macrophages with siRNA is often considered to be technically challenging, and, at present, few methodologies dedicated to the siRNA transfer to macrophages are available. Here, we present a protocol of using polyethyleneimine-coated superparamagnetic iron oxide nanoparticles (PEI-SPIONs) as a vehicle for the targeted delivery of siRNA to macrophages. PEI-SPIONs are capable of binding and completely condensing siRNA when the Fe:siRNA weight ratio reaches 4 and above. In vitro, these nanoparticles can efficiently deliver siRNA into primary macrophages, as well as into the macrophage-like RAW 264.7 cell line, without compromising cell viability at the optimal dose for transfection, and, ultimately, they induce siRNA-mediated target gene silencing. Apart from being used for in vitro siRNA transfection, PEI-SPIONs are also a promising tool for delivering siRNA to macrophages in vivo. In view of its combined features of magnetic property and gene-silencing ability, systemically administered PEI-SPION/siRNA particles are expected not only to modulate macrophage function but also to enable macrophages to be imaged and tracked. In essence, PEI-SPIONs represent a simple, safe, and effective nonviral platform for siRNA delivery to macrophages both in vitro and in vivo.

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

We describe a method of using polyethyleneimine (PEI)-coated superparamagnetic iron oxide nanoparticles for transfecting macrophages with siRNA. These nanoparticles can efficiently deliver siRNA to macrophages in vitro and in vivo and silence target gene expression.

Nanopartículas de óxido de ferro revestido de Polyethyleneimine como um veículo para a entrega de RNA de interferência pequeno para macrófagos In Vitro e In Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a ...

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow cytometry.

Isolamento de Adipócitos Viáveis e Fração Vascular Estromal do Tecido Adiposo Visceral Humano Adequado para Análise de RNA e Fenotipagem de Macrófagos

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These conditions include retinitis pigmentosa (RP) and advanced forms of age-related macular degeneration (AMD), such as geographic atrophy (GA). Together, these disorders represent a significant proportion of currently untreatable blindness globally. These unmet medical needs have generated heightened academic interest in developing methods of RPE replacement. Among the animal models commonly utilized for preclinical testing of therapeutics, the non-human primate (NHP) is the only animal model that has a macula. As it shares this anatomical similarity with the human eye, the NHP eye is an important and appropriate preclinical animal model for the development of advanced therapy medicinal products (ATMPs) such as RPE cell therapy.
This manuscript describes a method for the submacular transplantation of an RPE monolayer, cultured on a polyethylene terephthalate (PET) cell carrier, underneath the macula onto a surgically created RPE wound in immunosuppressed NHPs. The fovea-the central avascular portion of the macula-is the site of the greatest mechanical weakness during the transplantation. Foveal trauma will occur if the initial subretinal fluid injection generates an excessive force on the retina. Hence, slow injection under perfluorocarbon liquid (PFCL) vitreous tamponade is recommended with a dual-bore subretinal injection cannula at low intraocular pressure (IOP) settings to create a retinal bleb. 
Pretreatment with an intravitreal plasminogen injection to release parafoveal RPE-photoreceptor adhesions is also advised. These combined strategies can reduce the likelihood of foveal tears when compared to conventional techniques. The NHP is a key animal model in the preclinical phase of RPE cell therapy development. This protocol addresses the technical challenges associated with the delivery of RPE cellular therapy in the NHP eye.

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

Transplante de Epitélio de Pigmento De Retina em um modelo de primata não humano para doenças degenerativas da retina

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These ...