Questo lavoro è stato sostenuto dalla Generalitat de Catalunya (2005-SGR00437). Gli autori ringraziano a J. Blanco e F. Vidal per la loro supervisione e incoraggiamento, e J. Lucas per la sua assistenza tecnica.

I. Trattamento dei campioni e la fissazione delle cellule <ol><li> Lasciare il campione di seme in contenitori sterili a temperatura ambiente per 20 minuti fino a liquefazione. </li><li> Trasferire il campione in una provetta da centrifuga e spin è a 1000g per 5 minuti. </li><li> Rimuovere delicatamente e scartare il surnatante con una pipetta Pasteur. </li><li> Aggiungi una soluzione ipotonica (KCl, 0.075M) pre-riscaldato a 37 ° C, goccia a goccia, mescolando in un vortice di ottenere un volume finale di 10 ml. </li><li> Porre il tubo in un bagno d&#39;acqua a 37 ° C per 30 minuti. </li><li> Centrifugare a 1000g per 5 minuti. Eliminare attentamente il supernatante per decantazione, senza disturbare il pellet. </li><li> Dopo risospendere il pellet, aggiungere preparata Carnoy fissativo (3:1 metanolo: acido acetico) goccia a goccia mescolando in un vortice di ottenere un volume finale di 8 ml. </li><li> Ripetere i punti 6 e 7 come numero di volte necessario per ottenere una pallina bianca. </li><li> Aggiungere metanolo appena preparato: acido acetico (3:1) goccia a goccia per regolare il volume finale alla concentrazione cellulare necessarie per ottenere una buona diffusione delle cellule (nei casi con una piccola pallina, poche gocce sono sufficienti, mentre i campioni con una quantità elevata di spermatozoi recuperati, il volume finale può raggiungere 1-2 ml). Una diapositiva di prova può essere fatta facendo cadere il campione risospeso su uno scivolo ungreased ed esaminando al microscopio a contrasto di fase. Questo permetterà di controllare la dispersione cellulari ottenute e che permettono di correggere, se è necessario, in altre diapositive. </li><li> Per rendere le estensioni, caduta da una altezza minima di 40 cm due o tre gocce di sospensione cellulare al centro di diapositive unfrosted (precedentemente memorizzati in metanolo a -20 ° C a sgrassare loro). Per abilitare la posizione del campione in tutto il protocollo, segnare l&#39;area contenente l&#39;estensione sperma sul lato opposto della diapositiva con una matita diamante. </li><li> Conservare i campioni a -20 ° C per almeno 24 ore. Nota: L&#39;aggiunta del metanolo: acido acetico (3:1) goccia a goccia sotto agitazione è un passo molto importante per evitare la formazione di aggregati sperma. </li></ol> II. Decondensazione <ol><li> I vetrini conservati a -20 ° C deve essere scongelato per proseguire con il protocollo (lasciarli a temperatura ambiente). </li><li> Mettere il vetrino in due vaschette Coplin consecutivi con 2x salina-la soluzione di sodio citrato (2xSSC) per 3 minuti ciascuno. </li><li> Trasferire i vetrini attraverso una serie di lavaggi etanolo per 2 minuti in ogni vaschetta Coplin. Inizia con etanolo al 70%, seguite dal 90% e finire con il 100%. Asciugare il vetrino lasciandola a temperatura ambiente. </li><li> Incubare il vetrino in una soluzione ditiotreitolo (1,4-ditiotreitolo 5mm, 1% Triton X-100, 2-Amino-2-(idrossimetil) -1,3-propandiolo 50mm) a 37 ° C in incubatore a decondense della cromatina. Questo prodotto agisce rompendo i ponti disolfuro della protamine bobina che il DNA nel nucleo degli spermatozoi. Questo è un passo molto importante perché il DNA nel nucleo degli spermatozoi è molto compatta. Il tempo di incubazione delle diapositive in soluzione ditiotreitolo (DTT) deve essere regolata in base alla reattività del campione di questo prodotto. Di solito, questa volta è di circa 8 minuti, anche se può variare da 2 a 15 minuti. </li><li> Immediatamente, il trasferimento lo scivolo a due vasetti consecutivi Coplin con 2xSSC per 3 minuti in ogni Coplin. </li><li> Continuare mettendo la diapositiva con una serie di lavaggi etanolo per 2 minuti in ogni barattolo Coplin (70%, 90% e il 100% di etanolo). Lasciate che il vetrino ad asciugare a temperatura ambiente. Nota: l&#39;esposizione eccessiva al digitale terrestre porterebbe a disperdere i segnali FISH al termine della procedura, mentre una esposizione troppo breve si tradurrebbe in una mancanza di alcuni segnali. </li></ol> III. Ibridazione <ol><li> Denaturare il DNA dello sperma incubando il vetrino in una vaschetta Coplin con la soluzione formammide (70% Formamide/2xSSC) a 73 ° C per 5 minuti. </li><li> Trasferire il vetrino attraverso soluzioni di etanolo per 1 minuto per Coplin vaso (inizio con il 70% di etanolo, 85% e finire con il 100%). Lasciare il vetrino ad asciugare a temperatura ambiente. </li><li> Aggiungere direttamente 5 l del corrispondente pronti per l&#39;uso miscela sonda di una scivolata copertura 15x15 (Assay AneuVysion multi-colore del pannello della sonda: CEP 18/X/Y o LSI 13/21). </li><li> Come è mostrato nel video, posizionare con attenzione il vetrino sul vetrino di mettere insieme la regione di destinazione e la miscela della sonda. Sigillarlo con mastice. </li><li> Mettere il vetrino in una pre-riscaldato a 37 ° C camera di ibridazione (HYBrite ™) e incubare è a 37 ° C per 6-24 ore. Nota: considerando che la denaturazione del campione di sperma è obbligatoria, la necessità di denaturazione delle sonde è determinato dal produttore. Le miscele di sonda utilizzata in questo protocollo sono pronti all&#39;uso e, in questo caso, denaturazione della sonda non è necessaria. Il volume della miscela sonda varierà a seconda delle dimensioni dell&#39;area ibridato. </li></ol> IV. Lavapost-ibridazione <ol><li> Estrarre il vetrino dalla camera di ibridazione. </li><li> Rimuovere il mastice ed estrarre delicatamente la polizza di copertura scorrevole leggermente di lato. </li><li> Per eliminare i segnali di ibridazione aspecifica posto il vetrino per 2 minuti in una vaschetta Coplin con 0.4xSSC/0.3% NP-40 pre-riscaldato a 73 ° C in un bagno d&#39;acqua. </li><li> Trasferire i vetrini in una soluzione 2xSSC/0.1% NP-40 lavare a temperatura ambiente per 1 minuto. Lasciate che il vetrino ad asciugare a temperatura ambiente. </li><li> Aggiungere un prodotto di contrasto alla regione bersaglio (8 litri per una copertura antiscivolo 18x18). Il prodotto più comune utilizzato è 4 &#39;,6-diamidino-2-fenilindolo (DAPI, Vysis Inc.). </li><li> Come è mostrato nel video, la regione delle ibridato con un vetrino (per preservare l&#39;ibridazione la polizza di copertura può essere sigillato con smalto). </li><li> Conservare i vetrini ibridato a -20 ° C al buio fino a quando la loro analisi prospettiva. In queste condizioni le diapositive possono essere conservati per un massimo di 12 mesi senza perdita significativa dell&#39;intensità del segnale di fluorescenza. Nota: Una temperatura più alta o un tempo di lavaggio eccessive può provocare l&#39;eliminazione di alcuni segnali, mentre il contrario non sarebbe lavare ibridazioni sonda aspecifici. Il volume del prodotto di contrasto aggiunto varierà a seconda delle dimensioni dell&#39;area di ibridazione di coprire. </li></ol> V. visualizzazione La preparazione può essere valutato sotto un microscopio a epifluorescenza dotato di una tripla banda filtro per DAPI / Texas Red / FITC e single-band filtri per Aqua, FITC e Texas Red. Criteri di valutazione standard devono essere seguite per la corretta valutazione dei nuclei dello sperma 1. La preparazione ibridato con sonde per i cromosomi X, Y e 18 dovrebbe mostrare segnali per questi tre cromosomi. Ogni spermatozoi normali devono mostrare un segnale blu (corrispondente al cromosoma 18), e un segnale verde (X-cuscinetto spermatozoi) o di un segnale rosso (Y-cuscinetto spermatozoi). Nella preparazione ibridato con sonde per i cromosomi 13 e 21, un segnale verde per il cromosoma 13 e un segnale rosso per cromosoma 21 dovrebbero essere distinti in ogni spermatozoi normali.

<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.

Questo protocollo descrive come trattare i campioni di sperma umano per ottenere preparazioni FISH spermatozoi. Utilizzando questo protocollo è possibile analizzare le anomalie cromosomiche nei gameti maschili. Spermatozoi di screening aneuploidia ha applicazioni sia nel contesto della ricerca di base e nei consigli riproduttiva dato ai maschi infertili. Anche se nella diagnosi clinica dei cromosomi più studiati sono X, Y, 13, 18 e 21, altre sonde commerciali per una vasta gamma di cromosomi e loci sono disponibili e possono essere utilizzati anche in questi esperimenti.

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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

Aneuploidie sono le più frequenti anomalie cromosomiche negli esseri umani. La maggior parte di queste anomalie derivare da errori durante il processo meiotico gametogenic nei genitori. Nei maschi umani, questi errori possono portare alla produzione di spermatozoi con anomalie cromosomiche numeriche che rappresentano un aumento del rischio di trasmissione di queste anomalie alla prole. Per questo motivo, la tecnica di ibridazione in situ fluorescente (FISH) su nuclei degli spermatozoi è diventato un protocollo ampiamente adottato nel contesto della diagnosi clinica. Questa pratica fornisce una stima della frequenza di anomalie cromosomiche numeriche nei gameti dei pazienti che cercano di consulenza genetica riproduttiva. Ad oggi, i cromosomi più frequentemente inclusi nell&#39;analisi dello sperma FISH sono cromosomi X, Y, 13, 18 e 21. Questo video-articolo descrive, passo dopo passo, come elaborare e fissare un campione di sperma umano, come decondense e denaturare la cromatina spermatica, come procedere per ottenere preparazioni FISH spermatozoi, e come visualizzare i risultati al microscopio. Note particolari delle operazioni più rilevanti sono dati per ottenere i migliori risultati.

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

Questo video-articolo descrive, passo dopo passo, come elaborare un campione di sperma per raggiungere una buona qualità di fluorescenza<em&gt; In situ</emIbridazione&gt; sugli spermatozoi umani. Le preparazioni ottenute possono essere utilizzate per lo screening aneuploidia nel contesto della diagnosi clinica.

Fluorescenza In situ</emIbridazione> (FISH) Protocollo di sperma umano

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 Views.  Universitat Autónoma de Barcelona. Questo video-articolo descrive, passo dopo passo, come elaborare un campione di sperma per raggiungere una buona qualità di fluorescenza In situ sugli spermatozoi umani. Le preparazioni ottenute possono essere utilizzate per lo screening aneuploidia nel contesto della diagnosi clinica.

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.

Non radioattivo In situ Protocollo di ibridazione applicabile per la Norvegia Abete e una serie di specie di piante

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

Ricerca

Biologia

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.

Tecniche di Imaging Ca 2 + Segnalazione di sperma umano

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.

Rilevamento di RNA virale per fluorescenza<em&gt; In situ</em&gt; Hybridization (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.

Analisi di Single-cell trascrizione del gene per l&#39;RNA fluorescente<em&gt; In Situ</em&gt; Ibridazione (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.

Visualizzazione e l&#39;analisi di molecole di mRNA mediante fluorescenza<em&gt; In Situ</em&gt; Ibridazione in<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.

DNA-RNA combinato fluorescente<em&gt; In situ</em&gt; Ibridazione (FISH) per studiare inattivazione del cromosoma X nelle cellule staminali embrionali di topo differenziata Femminile

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.

Metodo semplice per DNA fluorescenza<em&gt; In Situ</em&gt; Ibridazione di cromosomi Squashed

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).

Osservazione e quantificazione dei telomeri e sequenze ripetitive Usando fluorescenza<em&gt; In Situ</em&gt; Ibridazione (FISH) con sonde PNA in<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.

Metodo per l'etichettatura di trascrizioni in cellule individuali Escherichia coli per singola molecola di fluorescenza In Situ gli esperimenti di ibridazione

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.

Valutazione di sperma di pesce utilizzando Software e dispositivi di raffreddamento

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

Fluorescenza In situ (FISH) Protocollo di sperma umano