Este trabajo fue apoyado por la Generalitat de Catalunya (2005-SGR00437). Los autores agradecen a J. Blanco y F. Vidal para su supervisión y estímulo, y Lucas J. por su asistencia técnica.

I. tratamiento de las muestras y la fijación de células <ol><li> Deja la muestra de semen en recipientes estériles a temperatura ambiente durante 20 minutos hasta la licuefacción. </li><li> Transferir la muestra a un tubo de centrífuga y girar a 1000 g durante 5 minutos. </li><li> Retire con cuidado y descartar el sobrenadante con una pipeta Pasteur. </li><li> Añadir una solución hipotónica (KCl, 0.075M) pre-calentado a 37 ° C, gota a gota, mientras se mezcla en un vórtice de obtener un volumen final de 10 ml. </li><li> Colocar el tubo en un baño de agua a 37 ° C durante 30 minutos. </li><li> Centrifugar a 1000 g durante 5 minutos. Elimine con cuidado el sobrenadante por decantación, sin perturbar el precipitado. </li><li> Después de resuspender el pellet, añada recién preparada fijador de Carnoy (3:1 metanol: ácido acético) gota a gota mientras se mezcla en un vórtice de obtener un volumen final de 8 ml. </li><li> Repetir los puntos 6 y 7 tantas veces como sea necesario para obtener una bolita blanca. </li><li> Añadir metanol recién preparada: ácido acético (3:1) gota a gota para ajustar el volumen final a la concentración celular requerida para la obtención de células buena difusión (en los casos con una pequeña bolita, unas cuantas gotas será suficiente mientras que las muestras con una gran cantidad de los espermatozoides recuperados, el volumen final puede llegar a 1-2 ml). Una diapositiva de prueba se puede hacer dejando caer la muestra se resuspendió en una diapositiva sin engrasar y examinarla bajo el microscopio de contraste de fase. Esto le permitirá comprobar la dispersión celular obtenido y que permita corregir, si es necesario, en una presentación de más. </li><li> Para hacer las extensiones, la caída de una altura mínima de 40 cm de dos o tres gotas de la suspensión celular en el centro de las diapositivas no congelado (previamente almacenados en metanol a -20 ° C para desengrasar). Para permitir la ubicación de la muestra durante todo el protocolo, marque el área que contiene la extensión de espermatozoides en el lado opuesto de la diapositiva con un lápiz de diamante. </li><li> Tienda de las diapositivas a -20 ° C durante al menos 24 horas. Nota: La adición de metanol: ácido acético (3:1) gota a gota, mientras que la mezcla es un paso muy importante para evitar la formación de agregados de espermatozoides. </li></ol> II. Descondensación <ol><li> Las diapositivas almacenadas a -20 ° C debe ser descongelado para continuar con el protocolo (dejarlos a temperatura ambiente). </li><li> Colocar el portaobjetos en dos frascos de Coplin consecutivos con una solución de 2x citrato salino-sódico (2XSSC) durante 3 minutos cada uno. </li><li> La transferencia de la diapositiva a través de una serie de lavados de etanol durante 2 minutos en cada frasco de Coplin. Comience con etanol al 70%, sigue en un 90% y terminar con un 100%. Secar el portaobjetos dejando a temperatura ambiente. </li><li> Incubar el portaobjetos en la solución de ditiotreitol (1,4-ditiotreitol 5 mM, 1% Triton X-100, 2-amino-2-(hidroximetil) -1,3-propanodiol 50 mm) a 37 ° C en la incubadora para decondense la cromatina. Este producto actúa rompiendo los puentes disulfuro de las protaminas que la bobina del ADN en el núcleo de los espermatozoides. Este es un paso muy importante porque el ADN en el núcleo del espermatozoide está muy compactado. El tiempo de incubación de los portaobjetos en solución de ditiotreitol (DTT) debe ajustarse de acuerdo a la reactividad de la muestra de este producto. Por lo general, esta vez es de alrededor de 8 minutos, aunque puede variar de 2 a 15 minutos. </li><li> Inmediatamente, la transferencia de la diapositiva a dos jarras Coplin consecutivos con 2XSSC durante 3 minutos en cada uno de Coplin. </li><li> Continúe colocando la diapositiva a través de una serie de lavados de etanol durante 2 minutos en cada frasco de Coplin (70%, 90% y el 100% de etanol). Dejó caer a secar a temperatura ambiente. Nota: La exposición excesiva a la TDT se traduciría en dispersar a las señales de FISH en el final del procedimiento, mientras que una exposición muy corta podría resultar en una falta de algunas señales. </li></ol> III. Hibridación <ol><li> Desnaturalizar el ADN de los espermatozoides mediante la incubación de la diapositiva en un frasco Coplin con una solución de formamida (70% Formamide/2xSSC) a 73 ° C durante 5 minutos. </li><li> La transferencia de la diapositiva a través de soluciones de etanol durante 1 minuto por cada frasco de Coplin (empezar con etanol al 70%, 85% y terminar con un 100%). Salga de la diapositiva a secar a temperatura ambiente. </li><li> Añadir directamente de 5 l de la correspondiente lista para usar mezcla de sondas de una hoja de cubierta de 15x15 (Ensayo AneuVysion Multi-color del panel de la sonda: CEP 18/X/Y o 13/21 LSI). </li><li> Como se muestra en el video, se coloca cuidadosamente la diapositiva en la hoja de la cubierta de armar la región de destino y la mezcla de la sonda. Sellarlo con cemento de goma. </li><li> Colocar la placa en un 37 precalentado ° C cámara de hibridación (HYBrite ™) y se incuba a 37 ° C durante 6-24 horas. Nota: Considerando que la desnaturalización de la muestra de esperma es obligatorio, la necesidad de la desnaturalización de las sondas está determinado por el fabricante. Las mezclas de sonda utilizada en este protocolo están listos para usar y, en este caso, la desnaturalización de la sonda no es necesario. El volumen de la mezcla de la sonda varía según el tamaño de la zona de hibridación. </li></ol> IV. Lavapost-hibridación <ol><li> Sacar el portaobjetos de la cámara de hibridación. </li><li> Quitar el cemento de caucho y tire con cuidado la hoja de cubierta deslizante suavemente a un lado. </li><li> Para eliminar las señales de hibridación no específica el lugar de la diapositiva durante 2 minutos en un frasco Coplin con 0.4xSSC/0.3% NP-40 pre-calentado a 73 ° C en un baño de agua. </li><li> Transferir el portaobjetos a una solución 2xSSC/0.1% NP-40 de lavado a temperatura ambiente durante 1 minuto. Dejó caer a secar a temperatura ambiente. </li><li> Añadir un producto contratinción de la región de destino (8 l de una hoja de cubierta 18x18). El producto más utilizado es de 4 &#39;,6-diamidino-2-phenylindole (DAPI, Vysis Inc.). </li><li> Como se muestra en el vídeo, la cobertura de la región hibridada con una hoja de cubierta (para preservar la hibridación puede ser la hoja de la cubierta sellada con esmalte de uñas). </li><li> Almacenar los portaobjetos hibridados a -20 ° C en la oscuridad hasta su análisis la perspectiva. En estas condiciones, las diapositivas se pueden almacenar hasta 12 meses sin pérdida significativa en la intensidad de la señal de fluorescencia. Nota: Una temperatura más alta o un tiempo de lavado excesivo puede resultar en la eliminación de algunas señales, mientras que lo contrario no se lave las hibridaciones inespecíficas de la sonda. El volumen del producto contratinción añadido variará según el tamaño de la zona de hibridación de principio a fin. </li></ol> V. Visualización Las preparaciones pueden ser evaluadas bajo un microscopio de epifluorescencia equipado con un filtro de triple banda de DAPI / Texas Red / FITC y la banda de un solo filtro de Aqua, FITC y Red Texas. Los criterios estándar de evaluación se deben seguir para la correcta evaluación de los núcleos de espermatozoides 1. La preparación de hibridación con sondas para los cromosomas X, Y y 18 debe mostrar señales de estos tres cromosomas. Todos los espermatozoides normales debe mostrar una señal de color azul (que corresponde al cromosoma 18), y una luz verde (X portador de espermatozoides) o una señal en rojo (de rodamientos espermatozoides). En la preparación de hibridación con sondas para los cromosomas 13 y 21, una señal verde para el cromosoma 13 y una señal en rojo para el cromosoma 21, ha de distinguirse en todos los espermatozoides normales.

<ol>
	<li>Blanco, J., Egozcue, J., Vidal, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+chromosome+21+disomy+in+human+spermatozoa+as+determined+by+fluorescent+in-situ+hybridization.">Incidence of chromosome 21 disomy in human spermatozoa as determined by fluorescent in-situ hybridization.</a> Hum. Reprod. 11, 722-726 (1996).</li></ol>

The authors have nothing to disclose.

Este protocolo describe la forma de procesar las muestras de semen humano para la obtención de preparaciones de esperma de pescado. El uso de este protocolo es posible analizar las anomalías cromosómicas en los gametos masculinos. Los espermatozoides de detección de aneuploidía tiene aplicaciones tanto en el contexto de la investigación básica y en el consejo reproductivo dado a los hombres infértiles. Aunque en el diagnóstico clínico de los cromosomas más estudiados son X, Y, 13, 18 y 21 de otras sondas comerciales para una amplia gama de los cromosomas y lugares están disponibles y también puede ser utilizado en estos experimentos.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>1,4-Dithiothreitol (DTT)</td>
<td>Material</td>
<td>Roche</td>
<td>10197777001</td>
<td>&nbsp;</td>
<tr>
<td>2-Amino-2-(hydroxymethyl)-1,3-propanediol</td>
<td>Material</td>
<td>Roche</td>
<td>10708976001</td>
<td>&nbsp;</td>
<tr>
<td>Acetic acid</td>
<td>Material</td>
<td>Merck</td>
<td>100.063</td>
<td>&nbsp;</td>
<tr>
<td>AneuVysion&reg; Multicolor DNA Probe Kit<ul style="padding:2px;"><li>20xSaline-Sodium Citrate</li><li>4,6-diamidino-2-phenylindole (DAPI II)</li><li>CEP 18/X/Y DNA probe</li><li>NP-40</li><li>LSI 13/21 DNA probe</li></ul></td>
<td>Material</td>
<td>Vysis Inc.</td>
<td>32-161075 <ul style="padding:2px;"><li>30-805850</li><li>30-804941 </li><li>30-171077</li><li>30-804820</li><li>30-171078</li></ul></td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge 5804R</td>
<td>Tool</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge tube</td>
<td>Material</td>
<td>Nunc</td>
<td>347856</td>
<td>&nbsp;</td>
<tr>
<td>Coplin jar (glass)</td>
<td>Material</td>
<td>Barloworld Scientific</td>
<td>Hellendahl (ZCT278)</td>
<td>&nbsp;</td>
<tr>
<td>Coplin jar (plastic)</td>
<td>Material</td>
<td>Deltalab</td>
<td>191087</td>
<td>&nbsp;</td>
<tr>
<td>Cover slips (15x15)</td>
<td>Material</td>
<td>Knittel gl&auml;ser</td>
<td>4600115</td>
<td>&nbsp;</td>
<tr>
<td>Cover slips (18x18)</td>
<td>Material</td>
<td>Knittel gl&auml;ser</td>
<td>4600118</td>
<td>&nbsp;</td>
<tr>
<td>Diamond pencil</td>
<td>Material</td>
<td>Hammacher</td>
<td>335117010</td>
<td>&nbsp;</td>
<tr>
<td>Ethanol</td>
<td>Material</td>
<td>Merck</td>
<td>100.983</td>
<td>&nbsp;</td>
<tr>
<td>Formamide</td>
<td>Material</td>
<td>Roche</td>
<td>11814320001</td>
<td>&nbsp;</td>
<tr>
<td>Freezer</td>
<td>Tool</td>
<td>Zanussi</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Gloves</td>
<td>Material</td>
<td>Sanyc</td>
<td>101/3300</td>
<td>&nbsp;</td>
<tr>
<td>HYBrite&trade;</td>
<td>Tool</td>
<td>Vysis Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Immersion Oil</td>
<td>Material</td>
<td>Olympus</td>
<td>35505</td>
<td>&nbsp;</td>
<tr>
<td>Incubator</td>
<td>Tool</td>
<td>Heraeus</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Methanol</td>
<td>Material</td>
<td>Merck</td>
<td>106.009</td>
<td>&nbsp;</td>
<tr>
<td>Micropipette</td>
<td>Material</td>
<td>Labsystems</td>
<td>Finnpipette (4500000)</td>
<td>&nbsp;</td>
<tr>
<td>Micropipette replacement tip</td>
<td>Material</td>
<td>Daslab</td>
<td>16-2001</td>
<td>&nbsp;</td>
<tr>
<td>Nail varnish</td>
<td>Material</td>
<td>Quo-cosmetics</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Olympus BX60 epifluorescence microscope</td>
<td>Tool</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>Equipped with a triple-band filter for DAPI/Texas Red/FITC and single-band filters for Aqua, FITC and Texas Red.</td>
</tr>
<tr>
<td>Pasteur Pipette</td>
<td>Material</td>
<td>Rubilabor</td>
<td>211.0230</td>
<td>&nbsp;</td>
<tr>
<td>Phase contrast microscope</td>
<td>Tool</td>
<td>Nikon Diaphot</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Plastic pipette (3ml)</td>
<td>Material</td>
<td>Deltalab</td>
<td>200007</td>
<td>&nbsp;</td>
<tr>
<td>Potassium chloride (KCl)</td>
<td>Material</td>
<td>Fluka</td>
<td>60128</td>
<td>&nbsp;</td>
<tr>
<td>Rubber cement</td>
<td>Material</td>
<td>Best-test</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Slides</td>
<td>Material</td>
<td>Knittel gl&auml;ser</td>
<td>4520022</td>
<td>&nbsp;</td>
<tr>
<td>Slide Saver Boxes</td>
<td>Material</td>
<td>Deltalab</td>
<td>19276.1</td>
<td>&nbsp;</td>
<tr>
<td>Sterile container</td>
<td>Material</td>
<td>Deltalab</td>
<td>409726</td>
<td>&nbsp;</td>
<tr>
<td>Syringe</td>
<td>Material</td>
<td>PentaFerte</td>
<td>002022300</td>
<td>&nbsp;</td>
<tr>
<td>Thermometer</td>
<td>Material</td>
<td>Comark Instruments, Inc</td>
<td>KM12</td>
<td>&nbsp;</td>
<tr>
<td>Tri-distilled water</td>
<td>Material</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>Material</td>
<td>Sigma</td>
<td>9002-93-1</td>
<td>&nbsp;</td>
<tr>
<td>Tweezers</td>
<td>Material</td>
<td>B/Braun</td>
<td>Aesculap (BD224R)</td>
<td>&nbsp;</td>
<tr>
<td>Vertical laminar flow hood</td>
<td>Tool</td>
<td>Burdinola</td>
<td>OR-ST 1500</td>
<td>&nbsp;</td>
<tr>
<td>Vortex mixer</td>
<td>Tool</td>
<td>Fisher Scientific</td>
<td>FB15012</td>
<td>&nbsp;</td>
<tr>
<td>Water bath</td>
<td>Tool</td>
<td>Raypa&reg;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

fluorescence in situ hybridization (fish) protocol in human sperm

Aneuploidías son las anomalías cromosómicas más frecuentes en los seres humanos. La mayoría de estas anomalías el resultado de errores durante el proceso de meiosis gametogénico en los padres. En los machos humanos, estos errores pueden conducir a la producción de espermatozoides con anomalías cromosómicas numéricas que representan un mayor riesgo de transmisión de estas anomalías a la descendencia. Por esta razón, la técnica de hibridación in situ fluorescente (FISH) en los núcleos de los espermatozoides se ha convertido en un protocolo ampliamente incorporado en el contexto del diagnóstico clínico. Esta práctica proporciona una estimación de la frecuencia de anomalías cromosómicas numéricas en los gametos de los pacientes que buscan el consejo genético reproductivo. Hasta la fecha, los cromosomas más frecuentemente incluidos en el análisis de esperma de pescado son los cromosomas X, Y, 13, 18 y 21. Este vídeo-artículo describe, paso a paso, cómo procesar y fijar una muestra de semen humano, cómo decondense y desnaturalizar la cromatina de los espermatozoides, la forma de proceder para obtener preparaciones de esperma de pescado, y la forma de visualizar los resultados en el microscopio. Comentarios especiales de los pasos más relevantes se dan para alcanzar los mejores resultados.

Watch this Scientific Journal Video about Fluorescence in situ hybridization FISH Protocol in Human Sperm at JoVE.com

Fluorescence in situ hybridization FISH Protocol in Human Sperm

Este vídeo-artículo describe, paso a paso, cómo se procesa una muestra de semen para lograr una buena calidad de fluorescencia<em&gt; In situ</em&gt; Hibridación en los espermatozoides humanos. Los preparados obtenidos pueden ser utilizados para la detección de aneuploidía en el contexto del diagnóstico clínico.

Fluorescencia In situ</emHibridación> (FISH) Protocolo en el esperma humano

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

fluorescence-in-situ-hybridization-fish-protocol-in-human-sperm

Nanyang Technological University

Research

JoVE Journal

Biology

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

30.0K vistas.  Universitat Autónoma de Barcelona. Este vídeo-artículo describe, paso a paso, cómo se procesa una muestra de semen para lograr una buena calidad de fluorescencia In situ Hibridación en los espermatozoides humanos. Los preparados obtenidos pueden ser utilizados para la detección de aneuploidía en el contexto del diagnóstico clínico.

Video: Fluorescence in situ hybridization FISH Protocol in Human Sperm

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ hybridization, a technique used to localize cell specific mRNA expression. The in situ hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies). Here we present a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana and Brassica napus. The protocol worked equally well for the species and genes studied. AtAP3 and BnAP3 were observed in second and third whorl floral organs in A. thaliana and B. napus and DAL13 in microsporophylls of male cones from P. abies. For P. abies the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.
Anna Karlgren and Jenny Carlsson contributed equally to this study.
Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundstr&#246;m at Jens.Sundstrom@vbsg.slu.se

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

We describe a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including Norway spruce. With just a few adjustments, including altered RNase treatment and proteinase K concentration, the protocol may be used in studies of different tissues and species.

No radiactivo In situ La hibridación aplicable para el abeto rojo protocolo y una serie de especies de plantas

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of ...

Investigación

Biología

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

Técnicas de Imagen Ca 2 + Señalización en el esperma humano

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

La detección de ARN viral por fluorescencia<em&gt; In situ</em&gt; La hibridación (FISH)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

Análisis de la transcripción de genes de una sola célula de ARN fluorescente<em&gt; In Situ</em&gt; La hibridación (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualización y Análisis de las moléculas de ARNm mediante fluorescencia<em&gt; In Situ</em&gt; La hibridación en<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

ADN-ARN combinado fluorescente<em&gt; In situ</em&gt; La hibridación (FISH) para el Estudio de inactivación del cromosoma X en las células madre embrionarias de ratón Diferenciada Mujer

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

Método simple de ADN de fluorescencia<em&gt; In Situ</em&gt; Hibridación a los cromosomas aplastado

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly ...

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase or an alternative pathway, known as alternative lengthening of telomeres (ALT)1. Recently, C. elegans has emerged as a multicellular model organism for the study of telomere and ALT2. Visualization of repetitive sequences in the genome is critical in understanding the biology of telomeres. While telomere length can be measured by telomere restriction fragment assay or quantitative PCR, these methods only provide the averaged telomere length. On the contrary, fluorescence in situ hybridization (FISH) can provide the information of the individual telomeres in cells. Here, we provide protocols and representative results of the method to determine telomere length of C. elegans by fluorescent in situ hybridization. This method provides a simple, but powerful, in situ procedure that does not cause noticeable damage to morphology. By using fluorescently labeled peptide nucleic acid (PNA) and digoxigenin-dUTP-labeled probe, we were able to visualize two different repetitive sequences: telomere repeats and template of ALT (TALT) in C. elegans embryos and gonads.

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans

We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT).

La observación y cuantificación de los telómeros y las secuencias repetitivas usando fluorescencia<em&gt; In Situ</em&gt; La hibridación (FISH) con sondas de PNA en<em&gt; Caenorhabditis elegans</em

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.

Método para el etiquetado de las transcripciones en los Escherichia coli las células para una sola molécula de la fluorescencia In Situ hibridación experimentos

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ ...

Fish Sperm Assessment Using Software and Cooling Devices

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems for sperm analysis were developed to measure several parameters and one of the most commonly measured is the sperm motility.
Initially, this computer technology was designed for mammalian species, although it can also be used for fish sperm analysis. Fish have specific features that can affect sperm assessment such as a short motility time after activation and, in some cases, adaptation to lower temperatures. Thus, it is necessary to modify both software and hardware components to make motility analysis more efficient for fish sperm analysis. For mammalian sperm, the heating plate is used to maintain optimal temperatures of spermatozoa. However, for some fish species, it is advantageous to use a lower temperature to prolong the duration of motility, since the sperm remain active for less than 2 min. Therefore, cooling devices are necessary to refrigerate samples at constant temperature over the time of analysis, including on the optical microscope. This protocol describes the analysis of fish sperm motility using software for sperm analysis and new cooling devices to optimize the results.

The present protocol describes a procedure of fish sperm assessment using computer-assisted sperm analysis and cooling devices. The software gives a rapid, accurate and quantitative analysis of fish sperm quality based on spermatozoa motility, which can be a useful tool in aquaculture to improve reproduction success.

Evaluación del esperma de pescado utilizando Software y equipos de refrigeración

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems ...

Fluorescencia In situ (FISH) Protocolo en el esperma humano