Este trabajo fue apoyado por el Fonds de Higiene de la Universidad Médica de Graz. Todos los sujetos dieron su consentimiento informado. La aprobación institucional del protocolo del estudio se obtuvo de la Universidad Médica de Graz.

1. Recogida de muestras <ol><li> Quitar fija expansores palatinos después de 4 meses de servicio intraoral. Utilizar el Sistema de NOLA campo seco para aislar a los aparatos antes de sacarlos (Figuras 1 y 2). </li><li> Utilice pinzas esterilizadas, guantes y bandejas para eliminar la contaminación sin necesidad de añadir expansores (Figuras 3 y 4). </li><li> Almacenar objetos en Sarstedt 120 ml viales a -20 ° C. Tome al laboratorio en hielo y el proceso dentro de las 24 horas. </li></ol> 2. Biofilm fijación <ol><li> Raspar escamas biofilm con un bisturí estéril, o cobrar piezas con una pinza estéril. </li><li> Añadir helada solución al 4% PFA hasta que la muestra está bien cubierta. </li><li> Incubar la mezcla a 4 ° C (no congelar) de 3 a 12 horas. Ya veces la fijación o la fijación de las temperaturas más altas pueden hacer que la envoltura celular de bacterias gram-negativos células menos permeable a las sondas de oligonucleótidos. </li><li> Retire la solución PFA y lavar con helado de PBS 1X. Repita este paso 2-3 veces a la remove residual PFA. </li><li> Resuspender la muestra en un vol. helado de PBS 1X y añadir vol 1. helada del 96% (v / v) de etanol. </li><li> Almacenar las muestras a -20 ° C. Las muestras fijadas de acuerdo con este protocolo se pueden almacenar por varios meses o años. </li></ol> 3. La deshidratación de las muestras fijas <ol><li> Aplicar l 50-30 de PFA-fijo material de la muestra a un portaobjetos de microscopio. </li><li> Seco a 46 ° C durante 15 min a temperatura ambiente o durante más tiempo. Descongele la lisozima y la formamida. </li><li> Añadir 250 l de lisozima (1mg/ml) a temperatura ambiente durante 10 minutos, lo que facilita la penetración de las sondas en las células por la destrucción parcial de las paredes celulares. </li><li> Dip caer en 50%, 80% y 96% (v / v) de etanol durante 3 minutos cada uno. El efecto deshidratante de la serie de la concentración de etanol va a desintegrar las membranas celulares. </li><li> Secar los portaobjetos a 46 ° C durante 10 min. </li></ol> 4. Hibridación in situ <ol><li> Prepare 1 ml detampón de hibridación fresca (ver Tabla 1 para la concentración). Las concentraciones de formamida utilizados en este estudio: 10% (EUBmix), el 20% (Bac303, institución de giro postal, MUT590) o 45% (LGCmix). </li><li> Descongele las soluciones sonda de oligonucleótidos. Sondas descongelado debe mantenerse en hielo y protegido de la luz. </li><li> Añadir 2 l de cada sonda de 200 l de tampón de hibridación, mezclar bien y aplicar la mezcla de la muestra deshidratada en un portaobjetos de microscopio. </li><li> Coloque un pedazo de papel de seda en una plaza una placa de Petri y verter el resto de tampón de hibridación en el papel de seda. </li><li> Inmediatamente colocar la placa horizontal en el plato y cerca del plato. Incubar en una estufa a 46 ° C durante 1-5 horas (90 minutos será suficiente en la mayoría de los casos). Las funciones de plato como una cámara de humedad evitando la evaporación de la solución de hibridación de la diapositiva. En particular, la evaporación de la formamida puede causar no específicos de la sonda unión a las células no deseadas. </li><li> Prepare 50 ml de tampón de lavado (ver Tabla 2 para concentrations). Prepare la solución de lavado en un tubo de 50 ml y de precalentamiento a 48 ° C en un baño de agua. La etapa de lavado se realiza a 48 ° C. </li><li> Retirar la cápsula con la tapa del horno de hibridación. Lávese inmediatamente el tampón de hibridación con un pequeño volumen de tampón de lavado pre-calentado, y la transferencia de la diapositiva en el buffer de lavado restante. </li><li> Coloque el tubo que contiene el tampón de lavado y deslice la espalda en el baño de agua y se incuba a 48 ° C durante 10-15 min. </li><li> Eliminar diapositiva del tubo y echar mano helada ddH 2 O durante 2-3 segundos para eliminar el tampón de lavado residual. </li><li> Deje secar al aire la diapositiva lo más rápido posible (el uso de aire comprimido se recomienda). De secado rápido se reducirá la sonda disociación. </li><li> Diapositivas seco se puede almacenar en la oscuridad a -20 ° C durante varias semanas sin una pérdida significativa de la sonda, confiere señal de fluorescencia. </li></ol> 5. Microscopía <ol><li> Después de FISH y lavado, aply dos gotas de cerca antifadent a los extremos izquierdo y derecho de una diapositiva (rodajas congeladas se debe calentar a temperatura ambiente antes de este paso). </li><li> Coloque una cubierta microscopio de deslizamiento en la parte superior y esperar hasta que el antifadent se ha extendido por toda la diapositiva. Tenga en cuenta que una cantidad excesiva de antifadent puede distorsionar la imagen del microscopio. </li><li> Observar las muestras con un microscopio láser confocal de barrido equipados con filtros adecuados o láser. Se utilizó un Leica TCS unidad (HCX PL APO/63x; NA 1.2). Los datos pueden ser analizados con el software como Imaris o AMIRA. </li><li> Diapositivas integrados en antifadent se puede almacenar a 4 ° C (no congelar) hasta 7 días antes de la sonda, confiere fluorescencia comienza a declinar. Por otra parte, la antifadent se puede quitar con ddH2O, y las diapositivas seco se puede almacenar a -20 ° C durante un período prolongado de tiempo. </li></ol> 6. Los resultados representativos: Raspado del biofilm de aparatos ortodóncicos fijos (Figura 5) los rendimientos de copos adecuado (Figura 6) que puede ser hibridado directamente sobre láminas de vidrio recubierto para microscopía. De esta manera, los diferentes grupos de bacterias orobiome se pueden identificar en sus recursos naturales, entorno tridimensional por rRNA bacteriano etiquetado con diferente marcado con sondas específicas (Figuras 7 y 8). En la figura 7, la biopelícula se tiñeron con EUBmix (verde, todas las bacterias) y LGCmix (amarillo, Firmicutes). Firmicutes aparecen en color verde, ya que estaban manchadas de amarillo y azul. En la Figura 8, la biopelícula se tiñeron con EUBmix (rojo, todas las bacterias), Bac303 (azul, Bacteroidetes) y instituciones de giro postal (amarillo, Porphyromonas gingivalis). Gingivalis se muestra en color blanco amarillento, como las tres sondas se unen a su ADN y la superposición de de los resultados de los colores en una señal de blanco. Las diferencias morfológicas entre los grupos de bacterias también pueden ser identificados (Figuras 9 y 10). Grandes grupos de bacterias cocoides se muestra en la Figura 9, donde la tinción se realizó con EUBmix (verde, todos los bacteria). Diferentes formas de las bacterias orales se visualizaron en la Figura 10, donde las bacterias cocoides y filamentosas se pueden distinguir por la tinción con EUBmix (rojo). Además, una típica seta-como la estructura del biofilm pueden ser procesados ​​a través del modelado 3D de los datos CLSM (Figuras 11 y 12) <a href="http://www.jove.com/files/ftp_upload/2967/Film1 Klug Santigli 01_2_JES_Web.mov">Haga clic aquí para ver una película de la modelación 3D.</a> <img alt="Figura 1" src="/files/ftp_upload/2967/2967fig1.jpg" /> Figura 1. Expansor palatino fijo in situ. <img alt="Figura 2" src="/files/ftp_upload/2967/2967fig2.jpg" /> Figura 2. Sistema de Nola campo seco. <img alt="Figura 3" src="/files/ftp_upload/2967/2967fig3.jpg" /> Figura 3. Pinzas esterilizadas, guantes y una bandeja. <img alt="Figura 4" src="/files/ftp_upload/2967/2967fig4.jpg" /> Figura 4. Eliminado de expansión. <img alt="Figura 5" src="/files/ftp_upload/2967/2967fig5.jpg" /> Figura 5. Raspado de biofilm con un bisturí estéril. <img alt="Figura 6" src="/files/ftp_upload/2967/2967fig6.jpg" /> Escamas de la figura 6. Resina directamente hibridizada en portaobjetos de vidrio recubierto. <img alt="Figura 7" src="/files/ftp_upload/2967/2967fig7.jpg" /> Figura 7 CLSM imagen:. Diferenciación de un grupo de bacterias específicas. Superposición de los datos 2D en 3D CLSM pila. Biofilm teñidas con EUBmix (verde, todas las bacterias) y LGCmix (amarillo, Firmicutes). <img alt="Figura 8" src="/files/ftp_upload/2967/2967fig8.jpg" /> Figura 8 CLSM imagen:. Diferenciación de un grupo de bacterias específicas. Superposición de los datos 2D en 3D CLSM pila. Biofilm teñidas con EUBmix (rojo, todas las bacterias), Bac303 (azul, Bacteroidetes) y instituciones de giro postal (amarillo, Porphyromonas gingivalis). <img alt="Figura 9" src="/files/ftp_upload/2967/2967fig9.jpg" /> Figura 9 CLSM imagen:. Diferenciación de las morfologías específicas. Superposición de los datos 2D en 3D CLSM pila. Biofilm teñidas con EUBmix (verde, todas las bacterias). Grandes grupos de bacterias cocoides (flechas). <img alt="Figura 10" src="/files/ftp_upload/2967/2967fig10.jpg" /> . Figura 10 imágenes CLSM: la diferenciación de las morfologías específicas. Superposición de los datos 2D en 3D CLSM pila. Biofilm teñidas con EUBmix (rojo, todas las bacterias). Cocoides (flecha abajo) y bacterias filamentosas (flecha arriba) se pueden distinguir. <img alt="Figura 11" src="/files/ftp_upload/2967/2967fig11.jpg" /> Figura 11. Estructura de hongos, vistas en 3D desde abajo. Las pilas de copos biofilm (raspado de la superficie de un aparato de ortodoncia), teñidas con EUBmix y procedimientosssed con Imaris (imagen CLSM). <img alt="Figura 11" src="/files/ftp_upload/2967/2967fig11.jpg" /> Figura 12. Estructura de hongos, vista lateral en 3D. Las pilas de copos biofilm (raspado de la superficie de un aparato de ortodoncia) elaborado con Imaris (imagen CLSM). <table border="1"><tbody><tr><td colspan="4"> La hibridación de amortiguación (200 l) </td></tr><tr><td> Concentración de formamida </td><td> 10% </td><td> 20% </td><td> 45% </td></tr><tr><td> 5 M NaCl </td><td> 36 </td><td> 36 </td><td> 36 </td></tr><tr><td> 1 M Tris-HCl </td><td> 4 </td><td> 4 </td><td> 4 </td></tr><tr><td> 2% de SDS </td><td> 1 </td><td> 1 </td><td> 1 </td></tr><tr><td> FA </td><td> 20 </td><td> 40 </td><td> 90 </td></tr><tr><td><strong&gt; DdH2O </td><td> 138 </td><td> 118 </td><td> 68 </td></tr><tr><td> Cualquier FISH sonda </td><td> 2 </td><td> 2 </td><td> 2 </td></tr></tbody></table> Tabla 1. Componentes de tampón de hibridación (concentraciones en l). <table border="1"><tbody><tr><td colspan="4"> Tampón de lavado (50 ml) </td></tr><tr><td> Concentración de formamida </td><td> 10% </td><td> 20% </td><td> 45% </td></tr><tr><td> 5 M NaCl </td><td> 4500 </td><td> 2150 </td><td> 300 </td></tr><tr><td> 1 M Tris-HCl </td><td> 1000 </td><td> 1000 </td><td> 1000 </td></tr><tr><td> EDTA 0,5 M </td><td> 0 </td><td> 500 </td><td> 500 </td></tr><tr><td> ddH2O </td><td> 44 500 </td><td> 46 350 </td&gt; <td> 48 200 </td></tr></tbody></table> Tabla 2. Constituyentes de tampón de lavado (concentraciones en l). Movie 1. <a href="http://www.jove.com/files/ftp_upload/2967/Film1 Klug Santigli 01_2_JES_Web.mov">Haga clic aquí para ver la película.</a>

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No hay conflictos de interés declarado.

El protocolo descrito en este documento es un enfoque muy práctico a los biofilms tinción recogida de materiales duros. Muestreo y la hibridación son los pasos más críticos. Durante el muestreo, se debe tener cuidado de que las rebanadas de un espesor adecuado se recogen para asegurar que la estructura del biofilm se mantiene intacta. Durante la hibridación, es esencial para evitar las fluctuaciones excesivas de temperatura, evitando así la unión no específica, o pérdida de unión, de las sondas de la etiqueta fluorescente. Este protocolo FISH es un método elegante para teñir biopelícula heterogénea normal directamente en placas de vidrio sin alterar su composición. Las diapositivas se pueden utilizar inmediatamente para microscopia, sin necesidad de mover la muestra una vez más. De esta manera, una mejora con respecto a la tinción de los tubos se consigue por la fuerza a menos de corte que se aplica a la biopelícula. Rodajas de&gt; 50 m de espesor se puede preparar e investigar de esta manera.

<table border="1">
	<tbody>
		<tr>
			<th>Oligonucleotides</th>
			<th>Target organism</th>
			<th>Fluorochrome</th>
		</tr>
		<tr>
			<td>EUB338 - 338III</td>
			<td>Eubacteria</td>
			<td>Cy3</td>
		</tr>
		<tr>
			<td>LGC354 A-C</td>
			<td>Firmicutes</td>
			<td>FITC</td>
		</tr>
		<tr>
			<td>Bac303</td>
			<td>Bacteroidetes</td>
			<td>Cy5</td>
		</tr>
		<tr>
			<td>Mut590</td>
			<td>Streptococcus mutans</td>
			<td>FITC</td>
		</tr>
		<tr>
			<td>POGI</td>
			<td>Porphyromonas gingivalis</td>
			<td>FITC</td>
		</tr>
	</tbody>
</table>
Table 3. Oligonucleotides (refer to probeBase13 for details).
<table border="1">
	<tbody>
		<tr>
			<th>Component</th>
			<th>Company</th>
			<th>Comments</th>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>&nbsp;</td>
			<td>4% solution in PBS</td>
		</tr>
		<tr>
			<td>1 x PBS</td>
			<td>&nbsp;</td>
			<td>3 x solution prepared</td>
		</tr>
		<tr>
			<td>ddH2O</td>
			<td>Millipore</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ethanol (100%)</td>
			<td>Merck</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Roth</td>
			<td>100 mg/ml stock solution</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Roth</td>
			<td>99.5% solution</td>
		</tr>
		<tr>
			<td>5 M NaCl</td>
			<td>Roth</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 M Tris-HCl</td>
			<td>Roth</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2% SDS</td>
			<td>Roth</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FISH probes (oligonucleotides)</td>
			<td>Invitrogen, Sigma</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ProLong Gold antifadent</td>
			<td>Invitrogen</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nail polish</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<th>Equipment</th>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo Scientific</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>IKA Tez</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>50 ml tubes</td>
			<td>Sarstedt</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polysine-coated slides</td>
			<td>Thermo Scientific</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
Table 4. Materials and equipment.

oral biofilm analysis of palatal expanders by fluorescence in-situ hybridization and confocal laser scanning microscopy

Microscopía de escaneo láser confocal (CLSM) de la biopelícula heterogénea naturales que hoy es facilitado por una amplia gama de técnicas de tinción, siendo uno de ellos hibridación in situ fluorescente (FISH). 1,2 Se realizó un estudio piloto en el que las muestras orales recogidas de biofilm fija aparatos de ortodoncia (expansores del paladar) fueron teñidas por los peces, con el objetivo de evaluar la organización tridimensional del biofilm natural y la acumulación de placa 3,4. PESCADO crea una oportunidad para teñir las células en su ambiente nativo de biofilm por el uso de 16S marcado con fluorescencia rRNA de metas de las sondas. 4-7,19 En comparación con otras técnicas, como el etiquetado de inmunofluorescencia, se trata de una técnica de bajo costo, el etiquetado preciso y sencillo para investigar diferentes grupos de bacterias en el biofilm de consorcios mixtos. 18,20 sondas general se utilizaron que se unen a las eubacterias ( EUB338 + + EUB338II EUB338III; adelante EUBmix), 80-10 Firmicutes (LGC354 AC;. LGCmix en adelante), 9,10 y Bacteroidetes (Bac303) 11 Además, las sondas de unión específico para Streptococcus mutans (MUT590) 12,13 y Porphyromonas gingivalis (institución de giro postal) 13,14 se utilizaron . La extrema dureza de los materiales de la superficie involucrada (de acero inoxidable y resina acrílica) nos obligó a buscar nuevas formas de preparación de la biopelícula. A medida que estos materiales de la superficie no puede ser fácilmente cortado con un cryotome, los distintos métodos de muestreo se analizaron para obtener biofilm bucal intacta. La más viable de estos enfoques se presenta en esta comunicación. Pequeñas escamas de la resina de biofilm de transporte de acrílico se desprendieron con un bisturí estéril, teniendo cuidado de no dañar la estructura del biofilm. Fórceps se utilizan para recoger la biopelícula de las superficies de acero. Una vez recogidas, las muestras fueron fijadas y se coloca directamente en Polysine diapositivas de vidrio recubierto. FISH se realiza directamente en las diapositivas con las sondas de mencionó arriba. Varios protocolos de peces se combinan y modifican para crear un nuevo protocolo que era fácil de manejar. 5,10,14,15 Posteriormente las muestras fueron analizadas por microscopía confocal de barrido láser. Conocido configuraciones 3,4,16,17 puede ser visualizado, incluyendo formaciones de estilo hongo y grupos de bacterias cocoides invadido por los canales. Además, la composición bacteriana de las estructuras típicas de biopelículas se analizaron y crearon imágenes en 2D y 3D.

Watch this Scientific Journal Video about Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy at JoVE.com

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Se presenta un protocolo para el análisis estructural y de composición del biofilm oral, natural de aparatos de ortodoncia con<em&gt; In situ</emHibridación&gt; (FISH) y la microscopía láser confocal de barrido (CLSM). Muestras orales de biopelículas se obtuvieron de expansores del paladar, el raspado de resina acrílica, copos de su superficie y someterlas a la transformación molecular.

Análisis de biofilm oral del paladar Expansores por fluorescencia de hibridación in situ y microscopía de escaneo láser confocal

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of ...

oral-biofilm-analysis-palatal-expanders-fluorescence-situ

Research

JoVE Journal

Medicine

15.9K vistas.  Medical University of Graz. Se presenta un protocolo para el análisis estructural y de composición del biofilm oral, natural de aparatos de ortodoncia con In situ (FISH) y la microscopía láser confocal de barrido (CLSM). Muestras orales de biopelículas se obtuvieron de expansores del paladar, el raspado de resina acrílica, copos de su superficie y someterlas a la transformación molecular.

Video: Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Probe-based Confocal Laser Endomicroscopy of the Urinary Tract: The Technique

Probe-based confocal laser endomicroscopy (CLE) is an emerging optical imaging technology that enables real-time in vivo microscopy of mucosal surfaces during standard endoscopy. With applications currently in the respiratory1 and gastrointestinal tracts,2-6 CLE has also been explored in the urinary tract for bladder cancer diagnosis.7-10 Cellular morphology and tissue microarchitecture can be resolved with micron scale resolution in real time, in addition to dynamic imaging of the normal and pathological vasculature.7
The probe-based CLE system (Cellvizio, Mauna Kea Technologies, France) consists of a reusable fiberoptic imaging probe coupled to a 488 nm laser scanning unit. The imaging probe is inserted in the working channels of standard flexible and rigid endoscopes. An endoscope-based CLE system (Optiscan, Australia), in which the confocal endomicroscopy functionality is integrated onto the endoscope, is also used in the gastrointestinal tract. Given the larger scope diameter, however, application in the urinary tract is currently limited to ex vivo use.11 Confocal image acquisition is done through direct contact of the imaging probe with the target tissue and recorded as video sequences. As in the gastrointestinal tract, endomicroscopy of the urinary tract requires an exogenenous contrast agent&mdash;most commonly fluorescein, which can be administered intravenously or intravesically. Intravesical administration is a well-established method to introduce pharmacological agents locally with minimal systemic toxicity that is unique to the urinary tract. Fluorescein rapidly stains the extracellular matrix and has an established safety profile.12 Imaging probes of various diameters enable compatibility with different caliber endoscopes. To date, 1.4 and 2.6 mm probes have been evaluated with flexible and rigid cystoscopy.10 Recent availability of a &lt; 1 mm imaging probe13 opens up the possibility of CLE in the upper urinary tract during ureteroscopy. Fluorescence cystoscopy (i.e. photodynamic diagnosis) and narrow band imaging are additional endoscope-based optical imaging modalities14 that can be combined with CLE to achieve multimodal imaging of the urinary tract. In the future, CLE may be coupled with molecular contrast agents such as fluorescently labeled peptides15 and antibodies for endoscopic imaging of disease processes with molecular specificity.

Probe-based confocal laser endomicroscopy enables real-time microscopy of the human urinary tract during cystoscopy, providing dynamic, intravital imaging of pathological states such as bladder cancer with cellular resolution. Endomicroscopy may augment the diagnostic accuracy of standard white light endoscopy and provide intraoperative image guidance to improve surgical resection.

Basada en sondeos endomicroscopía Láser Confocal del tracto urinario: la técnica

Probe-based  confocal laser endomicroscopy (CLE) is an emerging optical imaging technology  that enables real-time in vivo microscopy of mucosal surfaces ...

Investigación

Medicina

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA (Locked Nucleic Acid) probe. The probe was recognized by anti-DIG alkaline phosphatase antibodies and later developed using alkaline phosphatase substrate producing fluorescence signals. Here we utilized miRNA in situ hybridization (MISH) technique to analyze expression of miR-489 in tissues from breast cancer patients. This technique can detect the localization of miRNA of interest in individual tissue samples. This technique can be used to compare the expression of desired miRNA in tumor tissue with that in adjacent normal tissue and to identify the specific structures responsible for expressing this miRNA. This technique can be very useful in answering certain clinical questions, such as role of specific miRNA in the development of cancer. Our results indicate that mammary epithelial cells express significantly higher levels of miR-489 than adjacent tumor cells.

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

Here we present a protocol to detect miRNA expression in breast cancer patient samples using miRNA in situ hybridization.

Análisis clínico-patológica de los genes miARN expresión en el cáncer de mama mediante el uso de tejidos miARN<em&gt; In Situ</em&gt; La hibridación

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA ...

Oral Biofilm Sampling for Microbiome Analysis in Healthy Children

Oral biofilm and its molecular analysis provide a basis for investigating various dental research and clinical questions. Knowledge of biofilm composition leads to a better understanding of cariogenic and periopathogenic mechanisms. Microbial changes taking place in the oral cavity during childhood are of interest for several reasons. The evolution of the child oral microbiota and shifts in its composition need to be analyzed further to understand and possibly prevent the onset of disease. At the same time, advanced knowledge of the natural composition of oral biofilm is needed. Early stages of caries-free permanent dentition with healthy gums provide a widely unaffected subgingival habitat that can serve as an in situ baseline for studying features of oral health and disease. Analysis of children's oral biofilm during different stages in life is thus an important theme in the field. Modern molecular analysis methods can provide comprehensive information about the bacterial diversity of such biofilms. To enable microbiota data comparison, it is important to standardize each step in the procedure for molecular data generation. This procedure spans from clinical sampling, Next Generation Sequencing (NGS), bioinformatic data processing, to taxonomic interpretation. One of the most critical factors here is biofilm sampling. Sampling in children is even more challenging in particular due to limited space in subgingival areas. We thus focus on the use of paper points for subgingival sampling. This article provides a detailed protocol for oral biofilm sampling of the subgingival sulcus, the mucosa, and saliva in children.

Changes in the oral microbiome throughout childhood are of growing interest. Comparison of different microbiome studies reveals a lack of standardized sampling protocols. Limited space makes sampling the sound subgingival sulcus of children challenging. Paper point sampling is presented here in detail as the method of choice for this area.

Biofilm oral toma de muestras para análisis de microbioma en niños sanos

Oral biofilm and its molecular analysis provide a basis for investigating various dental research and clinical questions. Knowledge of biofilm composition ...

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy include photochemical cross-linking and tissue cross-linking (TXL) methods. Such methods for inducing mechanical tissue property changes are being employed to the cornea in corneal thinning (mechanically weakened) disorders such as keratoconus as well as the sclera in progressive myopia, where thinning and weakening of the posterior sclera occurs and likely contributes to axial elongation. The primary target proteins for such tissue strengthening are fibrillar collagens which constitute the great majority of dry weight proteins in the cornea and sclera. Fortuitously, fibrillar collagens are the main source of second harmonic generation signals in the tissue extracellular space. Therefore, modifications of the collagen proteins, such as those induced through cross-linking therapies, could potentially be detected and quantitated through the use of second harmonic generation microscopy (SHGM). Monitoring SHGM signals through the use of a laser scanning microscopy system coupled with an infrared excitation light source is an exciting modern imaging method that is enjoying widespread usage in the biomedical sciences. Thus, the present study was undertaken in order to evaluate the use of SHGM microscopy as a means to measure induced cross-linking effects in ex vivo rabbit sclera, following an injection of a chemical cross-linking agent into the sub-Tenon&#39;s space (sT), an injection approach that is standard practice for causing ocular anesthesia during ophthalmologic clinical procedures. The chemical cross-linking agent, sodium hydroxymethylglycinate (SMG), is from a class of cosmetic preservatives known as formaldehyde releasing agents (FARs). Scleral changes following reaction with SMG resulted in increases in SHG signals and correlated with shifts in thermal denaturation temperature, a standard method for evaluating induced tissue cross-linking effects.

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking TXL for Myopia

This protocol describes techniques for evaluating chemical cross-linking of the rabbit sclera using&#160;second harmonic generation imaging and differential scanning calorimetry.

Segunda generación armónica las señales en esclera de conejo como una herramienta para la evaluación del tejido terapéutico Cross-linking (TXL) para la miopía

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy ...

Oral Biofilm Formation on Different Materials for Dental Implants

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low microbial adhesion may reduce the prevalence and progression of peri-implant diseases. In view of the oral environment complexity and oral biofilm heterogeneity, microscopy techniques are needed that can enable a biofilm analysis of the surfaces of teeth and dental materials. This article describes a series of protocols implemented for comparing oral biofilm formation on titanium and ceramic materials for prosthetic abutments, as well as the methods involved in oral biofilms analyses at the morphological and cellular levels. The in situ model to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments as described in this study provides a satisfactory preservation of the 48 h biofilm, thereby demonstrating methodological adequacy. Multiphoton microscopy allows the analysis of an area representative of the biofilm formed on the test materials. In addition, the use of fluorophores and the processing of the images using multiphoton microscopy allows the analysis of the bacterial viability in a very heterogeneous population of microorganisms. The preparation of biological specimens for electron microscopy promotes the structural preservation of biofilm, images with good resolution, and no artifacts.

Here, we present a protocol to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments, including the analysis of bacterial cells viability and morphological characteristics. An in situ model associated with powerful microscopy techniques is used for the oral biofilm analysis.

Formación de Biofilm oral sobre diferentes materiales para implantes dentales

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low ...

Oral Health Assessment by Lay Personnel for Older Adults

Oral health is an often-undervalued contributor to overall health. The literature, however, underscores the myriad of systemic diseases influenced by oral health, including type II diabetes, heart disease, and atherosclerosis. Thus, assessments of oral health, called oral screenings, have a significant role in assessing risk of disease, managing disease, and even improving disease by oral care. Here we present a method to assess oral health quickly and consistently across time. The protocol is simple enough for non-oral health professionals such as students, family, and caregivers. Useful for any age of patient, the method is particularly key for older individuals who are often at risk of inflammation and chronic disease. Components of the method include existing oral health assessment scales and inventories, which are combined to produce a comprehensive assessment of oral health. Thus, oral characteristics assessed include intraoral and extraoral structures, soft and hard tissues, natural and artificial teeth, plaque, oral functions such as swallowing, and the impact this oral health status has on the patient&#39;s quality of life. Advantages of this method include its inclusion of measures and perceptions of both the observer and patient, and its ability to track changes in oral health over time. Results acquired are quantitative totals of questionnaire and oral screening items, which can be summed for an oral health status score. The scores of successive oral screenings can be used to track the progression of oral health across time and guide recommendations for both oral and overall health care.

The goal of this protocol is to enable non-dental professionals to assess oral health status for research or health-screening purposes. Aspects assessed include lips, tongue, soft and hard tissues, natural and artificial teeth, oral cleanliness, plaque, swallowing, and impact of oral health on quality of life.

Evaluación de salud oral por personal laicos para adultos mayores

Oral health is an often-undervalued contributor to overall health. The literature, however, underscores the myriad of systemic diseases influenced by oral ...

Visualizing Membrane Ruffle Formation using Scanning Electron Microscopy

Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may form spontaneously or in response to growth factors, inflammatory cytokines, and phorbol esters. Some of the membrane protrusions may reorganize into circular membrane ruffles that fuse at their distal margins and form cups that close and separate into the cytoplasm as large, heterogeneous vacuoles called macropinosomes. During the process, ruffles trap extracellular fluid and solutes that internalize within macropinosomes. High-resolution scanning electron microscopy (SEM) is a commonly used imaging technique to visualize and quantify membrane ruffle formation, circular protrusions, and closed macropinocytic cups on the cell surface. The following protocol describes the cell culture conditions, stimulation of the membrane ruffle formation in vitro, and how to fix, dehydrate, and prepare cells for imaging using SEM. Quantification of membrane ruffling, data normalization, and stimulators and inhibitors of membrane ruffle formation are also described. This method can help answer key questions about the role of macropinocytosis in physiological and pathological processes, investigate new targets that regulate membrane ruffle formation, and identify yet uncharacterized physiological stimulators as well as novel pharmacological inhibitors of macropinocytosis.

Macropinocytosis is a highly conserved endocytic process initiated by the formation of F-actin-rich sheet-like membrane projections, also known as membrane ruffles. Increased rate of macropinocytotic solute internalization has been implicated in various pathological conditions. This protocol presents a method to quantify membrane ruffle formation in vitro using scanning electron microscopy.

Visualización de la formación de volantes de membrana mediante microscopía electrónica de barrido

Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may ...

Interphase Fluorescence in situ Hybridization of Bone Marrow Smears of Multiple Myeloma

Fluorescence in situ hybridization (FISH) detection is an indispensable method in genetic risk stratification in multiple myeloma (MM), which is one of the most common hematological malignancies. The identifying characteristic of MM is accumulated malignant plasma cells in bone marrow. FISH reports for MM mainly focus on purified or identified clonal plasma cells, rather than all nucleated cells, by sorting with anti-CD138 magnetic beads or marking with cytoplasmic immunoglobulin light chain &#954; or &#955;. Bone marrow interphase nuclei are usually obtained from fresh bone marrow cells. However, satisfactory enrichment of plasma cell specimens requires large amounts of fresh heparin anti-coagulated bone marrow, which cannot be obtained in the case of difficult bone marrow extraction or a bone marrow dry tap. Herein, we establish a novel method to improve the success of FISH detection on stained or unstained bone marrow smears. Bone marrow smears are easier to obtain than anticoagulated bone marrow specimens.

Interphase Fluorescence in situ Hybridization of Bone Marrow Smears of Multiple Myeloma

Here, we present a protocol for improving the success of interphase fluorescence in situ hybridization detection on bone marrow smears from multiple myeloma patients.

Hibridación de fluorescencia in situ interfásica de frotis de médula ósea de mieloma múltiple

Fluorescence in situ hybridization (FISH) detection is an indispensable method in genetic risk stratification in multiple myeloma (MM), which is one of ...

Semi-Automated Planimetric Quantification of Dental Plaque Using an Intraoral Fluorescence Camera

Dental plaque accumulation is quantified using clinical indices or, otherwise, the planimetric plaque index (PPI),&#160;which measures the relative area of a tooth that is covered by plaque deposits. Compared to clinical indices, the PPI has a higher discriminatory power, but traditional planimetry is a time-consuming analysis, as the plaque-covered and clean tooth areas have to be determined manually for each image using image-processing software. Here, we present a method for the semi-automated planimetric quantification of dental plaque, which allows for the rapid processing of up to 1,000 images simultaneously. The method exploits the enhanced contrast between disclosed plaque, sound tooth surfaces, and soft tissues in fluorescence images acquired with an intraoral camera. Careful execution of the clinical procedures and accurate image acquisition are crucial steps for the successful semi-automated identification of the plaque-covered areas. The method is suitable for planimetry on sound facial and oral tooth surfaces, on most composite resin restorations, and on teeth with orthodontic brackets, but not on metallic restorations. Compared to traditional PPI recordings, semi-automated planimetry considerably reduces the amount of time spent on the analysis, as well as the subjective human input, thus increasing the reproducibility of planimetric measurements.

This study presents a semi-automated digital image analysis procedure for the planimetric quantification of disclosed dental plaque based on images acquired with an intraoral fluorescence camera. The method allows for the rapid and reliable quantification of dental plaque in the research environment.

Cuantificación planimétrica semiautomática de placa dental mediante una cámara de fluorescencia intraoral

Dental plaque accumulation is quantified using clinical indices or, otherwise, the planimetric plaque index (PPI),&#160;which measures the relative area ...

Captura de muestras de tejido de fibrosis submucosa oral para análisis espacial-ómico

Isolation of Cells with Morphological and Spatial Information from Oral Submucous Fibrosis Samples by Laser Capture Microdissection

Oral submucous fibrosis (OSF) is a common type of potentially malignant disorder in the oral cavity. The atrophy of epithelium and fibrosis of the lamina propria and the submucosa are often found on histopathological slides. Epithelial dysplasia, epithelial atrophy, and senescent fibroblasts have been proposed to be associated with the malignant transformation of OSF. However, because of the heterogeneity of potentially malignant oral disorders and oral squamous cell carcinoma, it is difficult to identify the specific molecular mechanisms of malignant transformation in OSF. Here, we present a method to obtain a small number of epithelial or mesenchymal cells carrying morphological data and spatial information by laser capture microdissection on formalin-fixed paraffin-embedded tissue slides. Using a microscope, we can precisely capture microscale (~500 cells) dysplastic or atrophic epithelial tissue and fibrotic subepithelial tissue. The extracted cells can be evaluated by genome or transcriptome sequencing to acquire genomic and transcriptomic data with morphological and spatial information. This approach removes the heterogeneity of bulk OSF tissue sequencing and the interference caused by cells in non-lesioned areas, allowing for precise spatial-omics analysis of OSF tissue.

Autor destacado: Desvelando los misterios de las posibles neoplasias malignas orales 

Laser capture microdissection of oral submucous fibrosis tissues allows for precise extraction of cells from histological regions of interest for the analysis of multi-omics data with morphological and spatial information.

Aislamiento de Células con Información Morfológica y Espacial a partir de Muestras de Fibrosis Submucosa Oral mediante Microdisección por Captura Láser

Oral submucous fibrosis (OSF) is a common type of potentially malignant disorder in the oral cavity. The atrophy of epithelium and fibrosis of the lamina ...