Ringrazio Jeannie T. Lee per la supervisione di questo progetto quando ero nel laboratorio di Lee e Tyler Broering per aver modificato il manoscritto. Questo lavoro è stato supportato dal Basil O'Connor Starter Scholar Award della March of Dimes Foundation e dal NIH Grant GM098605.

1.3D preparazione delle diapositive
<ol><li>Preparare i seguenti reagenti:<ol>
<li>Tampone CSK: NaCl da 100 mM, saccarosio da 300 mM, tubi da 10 mM, 3 mM MgCl2. Regolare il pH a 6,8 con 1 M NaOH. Conservare il buffer CSK a 4 °C.</li>
		<li>Tampone CSK con 0,5% Tritone X-100: Aggiungere 200 μl di Tritone X-100 a 40 ml di tampone CSK. Mescolare la soluzione utilizzando l'agitatore magnetico fino a quando Triton X-100 non viene completamente sciolto. Conservare il buffer CSK con 0,5% Tritone X-100 a 4 °C.</li>
		<li>4% Paraformaldeide (PFA)–PBS, pH 7.4: Sciogliere 4 g di PFA in circa 70-80 ml di acqua. Per accelerare il processo di dissoluzione, aggiungere circa 20 μl di 4 N NaOH e mantenere la soluzione in un bagno d'acqua a 60 °C fino a quando la PFA non si è completamente sciolta e la soluzione è trasparente. Questo processo richiede circa 1 ora. Dopo che la PFA si è completamente sciolta, aggiungere 10 ml di 10x PBS. Regolare il volume finale a 100 ml con acqua e raffreddare la soluzione a temperatura ambiente (25 °C). Utilizzare una soluzione preparata al momento per ogni nuovo esperimento. Nota: ottenere l'autorizzazione istituzionale per tutto il lavoro sugli animali e aderire alle linee guida pertinenti per la cura degli animali.</li>
	</ol>
</li>
	<li>Eutanasia dei topi maschi. Usando forcep e forbici, asporta il testicolo dal mouse. Posizionare il testicolo in PBS (o RPMI 1640). Dopo la rimozione del testicolo, eseguire rapidamente i seguenti passaggi 1.3-1.4 al fine di prevenire la degradazione dell'RNA.</li>
	<li>Rompono il testicolo su una piccola piastra di Petri e tolgono la tunica albuginea. Dipanare i tubuli seminiferi in PBS sul ghiaccio usando le forcep.</li>
	<li>Utilizzando le forcep, trasferire diversi tubuli (diversi pezzi di tubuli di circa 5-10 mm di lunghezza) in un pozzo di un piatto da 4 pozzetti contenente 500 ml di tampone CSK + 0,5% Tritone X-100 sul ghiaccio e incubare per 6 minuti. La sovraesposizione al buffer CSK può causare l'interruzione delle celle.</li>
	<li>Utilizzando le forcep, trasferire tutti i tubuli in un unico pozzo di un piatto da 4 pozzetti contenente il 4% di soluzione PFA-PBS. Questo passaggio può essere preformato senza stereomicroscopio, ma opzionalmente può essere utilizzato. Incubare per 10 minuti a temperatura ambiente. Inizia contemporaneamente a preparare 12 camere di citospino durante i periodi di incubazione.</li>
	<li>Utilizzando le forcep, trasferire tutti i tubuli in un pozzo di un piatto da 4 pozzetti contenente PBS. Incubare per 5 minuti a temperatura ambiente.</li>
	<li>Sul retro di uno scivolo di vetro, trasferire tutti i tubuli in 30 μl di PBS (la parte superiore dello scivolo deve essere evitata a causa della carica cationica). Usando le punte di due forcep, strappare i tubuli a pezzi. Tritare i tubuli in direzione orizzontale ritagliando i tubuli tra le punte di due forcep e tirando le forcep orizzontalmente. Continuare con questo passaggio per circa 10-20 secondi.</li>
	<li>Mescolare la sospensione tubazione utilizzando una pipetta P20.</li>
	<li>Trasferire la sospensione su un tubo di microcentrifugo, diluire con PBS a circa 1,3 ml. Applicare 100 μl di sospensione su ciascuna delle 12 camere di citospino (1,2 ml totali). In questo passaggio, la concentrazione di cellule non ha bisogno di essere determinata.</li>
	<li>Citospin i campioni a 2.000 giri/min per 10 minuti a temperatura ambiente. Dopo la citospinizzazione, scivoli asciutti sul banco di laboratorio per alcuni minuti a temperatura ambiente. Dopo che le diapositive sono state autorizzate ad asciugarsi completamente per alcuni minuti, passare al passaggio successivo.</li>
	<li>Utilizzando un barattolo coplin contenente PBS, lavare gli scivoli per 5 minuti a temperatura ambiente.</li>
	<li>Trasferire le diapositive in un barattolo di Coplin contenente il 70% di etanolo e attendere almeno 2 minuti prima dell'inizio dell'RNA FISH. Le diapositive sono stabili per alcune settimane con 70% di etanolo a 4 °C. Per la conservazione a lungo termine, disidratare le diapositive utilizzando il trattamento seriale con 80% e 100% di etanolo in barattoli Coplin per 2 minuti ciascuno e asciugare all'aria completamente disidratare le diapositive. Conservare a -80 °C in una slide box.</li>
</ol>2. Preparazione della sonda del DNA Cot-1 mediante priming casuale
<ol><li>Preparare i seguenti reagenti:<ol>
<li>Buffer di ibridazione: Mescolare i componenti in corso per preparare 900 ml di scorta stabilizzatrice di ibridazione; questa soluzione verrà utilizzata per rifornire le sonde. Questa scorta stabilizzatrice di ibridazione consente l'aggiunta del 10% del volume della soluzione inibitore della RNasi (Ribonucleoside Vanadyl Complex) prima dell'ibridazione.<table><tbody>
<tr>
<td>componente</td>
					<td>Importo (μl)</td>
					<td>Concentrazione finale</td>
				</tr>
<tr>
<td>Formamide</td>
					<td>500</td>
					<td>50% (v/v)</td>
				</tr>
<tr>
<td>50% di Dextran</td>
					<td>200</td>
					<td>10% (con)</td>
				</tr>
<tr>
<td>20x SSC</td>
					<td>100</td>
					<td>2x</td>
				</tr>
<tr>
<td>1% BSA</td>
					<td>100</td>
					<td>0.1%</td>
				</tr>
</tbody></table>Prima dell'ibridazione, aggiungere 1/10 vol di 200 mM ribonucleoside Vanadyl Complex o acqua alla scorta stabilizzatrice di ibridazione per abbinare la concentrazione finale. A -20 °C, questa miscela è stabile per più di un anno.</li>
		<li>20x SSC: Sciogliere 88,2 g di citrato di sodio e 175,3 g di NaCl in circa 800 ml di acqua. Regolare il pH a 7.0 utilizzando alcune gocce di 1 M HCl. Aggiungere acqua per regolare il volume finale a 1 L. Sterilizzare mediante autoclave. Dopo la preparazione, 20x SSC possono essere conservati a temperatura ambiente.</li>
		<li>50% (w/v) Dextran: Mescolare l'acqua e aggiungere lentamente 50 g di solfato di dextran. Continuare a mescolare durante la notte a temperatura ambiente. Aggiungere acqua per regolare il volume finale a 100 ml. Conservare a temperatura ambiente.</li>
	</ol>
</li>
	<li>Aggiungere i seguenti componenti elencati di seguito a un tubo PCR. Utilizzando una macchina PCR, mescolare e incubare la soluzione per 5 minuti a 95 °C.<table><tbody>
<tr>
<td>componente</td>
				<td align="center">Quantità per reazione (μl)</td>
				<td align="center">finale</td>
			</tr>
<tr>
<td>DNA Cot-1: 1 mg/ml</td>
				<td align="center">1</td>
				<td align="center">1 μg</td>
			</tr>
<tr>
<td>Primer random 9mer (dal kit)</td>
				<td align="center">10</td>
				<td align="center"></td>
			</tr>
<tr>
<td>Acqua</td>
				<td align="center">27</td>
				<td align="center"></td>
			</tr>
</tbody></table>
</li>
	<li>Centrifugare brevemente e posizionare sul ghiaccio.</li>
	<li>Aggiungere i componenti seguenti. Mescolare e incubare per 30 min a 37 °C utilizzando una macchina PCR. (Per i dettagli del kit utilizzato si veda la tabella dei reagenti e delle attrezzature).<table><tbody>
<tr>
<td>componente</td>
				<td align="center">Quantità per reazione (μl)</td>
				<td align="center">Concentrazione finale</td>
			</tr>
<tr>
<td>Miscela preparata a partire dal passaggio 2.2</td>
				<td align="center">38</td>
				<td align="center"></td>
			</tr>
<tr>
<td>Tampone nucleotidico 5x (dal kit)</td>
				<td align="center">9.2</td>
				<td align="center"></td>
			</tr>
<tr>
<td>Cy3-dUTP (1 mM)</td>
				<td align="center">0.8</td>
				<td align="center">16 μM</td>
			</tr>
<tr>
<td>Klenow (dal kit)</td>
				<td align="center">2</td>
				<td align="center"></td>
			</tr>
</tbody></table>
</li>
	<li>Aggiungere 2 μl di tampone di arresto (dal kit) per interrompere la reazione.</li>
	<li>Purificare la reazione interrotta utilizzando colonne di microspin.</li>
	<li>Aggiungere 50 μg (5 μl) di DNA dello sperma di aringa (50 volte superiore al DNA del modello Cot-1) alla frazione purificata.</li>
	<li>Misurare il volume totale utilizzando una pipetta. Aggiungere 0,7x volume di etanolo al 100% e volume 0,1x di acetato di sodio 3 M (pH 5.2), mescolare bene e centrifugare per 15 minuti alla velocità massima (17.000 x g) utilizzando un microcentrifugo a 4 °C.</li>
	<li>Rimuovere il liquido e aggiungere 200 μl di etanolo al 70%, mescolare bene e centrifugare per 5 minuti alla velocità massima a 4 °C.</li>
	<li>Rimuovere il liquido in eccesso. Asciugare il precipitato per 10-30 minuti a temperatura ambiente.</li>
	<li>Sciogliere il pellet in 20 μl di scorta stabilizzatrice di ibridazione. Poiché il pellet è spesso difficile da sciogliere, pipettare ripetutamente. Come opzione alternativa, un miscelatore a vortice può anche essere utilizzato per sciogliere in modo efficiente il pellet. Le sonde possono essere mantenute a -20 °C fino all'uso.</li>
</ol>3. Cot-1 RNA FISH
<ol><li>Aggiungere i componenti elencati di seguito a un tubo PCR. Utilizzando una macchina PCR, incubare per 10 min a 80 °C, seguito da una fase di preannealizzazione di circa 10-30 min a 42 °C. Conservare a 42 °C fino all'ibridazione. Ribonucleoside Vanadyl Complex è facoltativo; può essere sostituito con acqua.<table><tbody>
<tr>
<td>componente</td>
				<td align="center">Quantità per reazione (μl)</td>
				<td align="center">finale</td>
			</tr>
<tr>
<td>Sonda A DNA Cot-1 (50 ng/μl)</td>
				<td align="center">2</td>
				<td align="center">100 ng</td>
			</tr>
<tr>
<td>Complesso Di Vanadyl Ribonucleoside (200 mM)</td>
				<td align="center">2</td>
				<td align="center">20 mM</td>
			</tr>
<tr>
<td>Buffer di ibridazione</td>
				<td align="center">aggiunto a 20</td>
				<td align="center"></td>
			</tr>
</tbody></table>
</li>
	<li>Vetrini disidratati in un barattolo di Coplin contenente 80% di etanolo per 2 min, seguiti da un barattolo di Coplin contenente 100% di etanolo per 2 min. Scivoli asciutti completamente sulla panchina del laboratorio a temperatura ambiente.</li>
	<li>Preriscaldare in anticipo una camera a punta (riempita con 2-3 cm di acqua) in un incubatore a 42 °C. Posizionare la diapositiva sulla camera della casella di punta. Centrifugare brevemente le sonde preannealate e la pipetta direttamente sugli scivoli disidratati. Evitare di fare bolle. Coprire delicatamente lo scivolo con un foglietto di copertura.</li>
	<li>Ibridare per 6 ore fino a durante la notte (14-15 ore) a 42 °C.</li>
	<li>Preparare soluzioni di lavaggio in vasetti Coplin (due vasetti con 2x SSC e 50% formamide, e due vasetti con 2x SSC). Preriscaldare a 42 °C per 30 minuti in un bagno d'acqua.</li>
	<li>Utilizzare una lama per rimuovere lo slittamento del coperchio dal bordo dello scivolo. Evitare di graffiare i campioni. Lavare gli scivoli in un barattolo coplin contenente 2x SSC e 50% formamide per 5 min a 45 °C. Ripetere questo passaggio una volta.</li>
	<li>Lavare gli scivoli in un barattolo coplin contenente 2x SSC per 5 minuti a 45 °C. Ripetere questo passaggio una volta.</li>
	<li>Aggiungere 20 μl di supporti di montaggio DAPI alle diapositive e coprire con uno slittamento di copertura. Premere delicatamente il vetro di copertura per rimuovere i supporti di montaggio aggiuntivi. Blot supporti di montaggio extra con tessuto detergente. Le diapositive sono pronte per la valutazione della microscopia.</li>
</ol>

<ol>
	<li>Peters, A. H., Plug, A. W., van Vugt, M. J., de Boer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+drying-down+technique+for+the+spreading+of+mammalian+meiocytes+from+the+male+and+female+germline.">A drying-down technique for the spreading of mammalian meiocytes from the male and female germline.</a> Chromosome Res. 5, 66-68 (1997).</li><li>Namekawa, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postmeiotic+sex+chromatin+in+the+male+germline+of+mice.">Postmeiotic sex chromatin in the male germline of mice.</a> Curr Biol. 16, 660-667 (2006).</li><li>Namekawa, S. H., Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+nascent+RNA,+single-copy+DNA+and+protein+localization+by+immunoFISH+in+mouse+germ+cells+and+preimplantation+embryos.">Detection of nascent RNA, single-copy DNA and protein localization by immunoFISH in mouse germ cells and preimplantation embryos.</a> Nat Protoc. 6, 270-284 (2011).</li><li>Mahadevaiah, S. K., Costa, Y., Turner, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+RNA+FISH+to+study+gene+expression+during+mammalian+meiosis.">Using RNA FISH to study gene expression during mammalian meiosis.</a> Methods in molecular biology. 558, 433-444 (2009).</li><li>Hall, L. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ectopic+human+XIST+gene+can+induce+chromosome+inactivation+in+postdifferentiation+human+HT-1080+cells.">An ectopic human XIST gene can induce chromosome inactivation in postdifferentiation human HT-1080 cells.</a> Proc Natl Acad Sci U S A. 99, 8677-8682 (2002).</li><li>Huynh, K. D., Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inheritance+of+a+pre-inactivated+paternal+X+chromosome+in+early+mouse+embryos.">Inheritance of a pre-inactivated paternal X chromosome in early mouse embryos.</a> Nature. 426, 857-862 (2003).</li><li>Namekawa, S. H., VandeBerg, J. L., McCarrey, J. R., Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+chromosome+silencing+in+the+marsupial+male+germ+line.">Sex chromosome silencing in the marsupial male germ line.</a> Proceedings of the National Academy of Sciences of the United States of America. 104, 9730-9735 (2007).</li><li>Namekawa, S. H., Payer, B., Huynh, K. D., Jaenisch, R., Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-step+imprinted+X+inactivation:+repeat+versus+genic+silencing+in+the+mouse.">Two-step imprinted X inactivation: repeat versus genic silencing in the mouse.</a> Mol Cell Biol. 30, 3187-3205 (2010).</li><li>Ichijima, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MDC1+directs+chromosome-wide+silencing+of+the+sex+chromosomes+in+male+germ+cells.">MDC1 directs chromosome-wide silencing of the sex chromosomes in male germ cells.</a> Genes Dev. 25, 959-971 (2011).</li><li>Sin, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNF8+regulates+active+epigenetic+modifications+and+escape+gene+activation+from+inactive+sex+chromosomes+in+post-meiotic+spermatids.">RNF8 regulates active epigenetic modifications and escape gene activation from inactive sex chromosomes in post-meiotic spermatids.</a> Genes Dev. 26, 2737-2748 (2012).</li></ol>

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Nella preparazione della diapositiva 3D, la fase di permeabilizzazione precede la fase di fissazione. Questi passaggi vengono eseguiti direttamente su tubuli seminiferi, preservando così la struttura tridimensionale della cromatina. Un'opzione alternativa per preservare la struttura della cromatina per RNA / DNA FISH è eseguire contemporaneamente la fase di permeabilizzazione e la fase di fissazione. Questa tecnica alternativa è stata eseguita nelle cellule germinali marsupiali e negli embrioni di preimpianto del...

Nei teste, le cellule germinali si differenziano dalla spermatogonia diploide attraverso la meiosi in spermatozoi maturi e aploidi. Durante questo processo, la struttura della cromatina nucleare delle cellule germinali viene continuamente e dinamicamente rimodellata. Le crepa superficiali sono comunemente utilizzate per l'esame citologico delle singole cellule spermatogeniche. Un metodo prevalente di diffusione superficiale utilizza un trattamento ipotonico con cui le singole cellule spermatogeniche vengono diffuse e appiattite1. Queste condizioni sono ottimali per l'analisi dettagliata dei cromosomi meiotici. Le caratteristiche cromosomiche come lo stato sinaptico e i focolai di ricombinazione sono facilmente osservabili usando questo metodo. Tuttavia, il trattamento ipotonico interrompe l'architettura della cromatina subnucleare, quindi questa tecnica non è adatta per l'analisi strutturale dei nuclei. Di conseguenza, è stato progettato un metodo migliorato per preservare la struttura tridimensionale della cromatina delle cellule germinali testicolari. Questo metodo è stato definito metodo di scorrimento tridimensionale (3D) (Figura 1). Il metodo di scorrimento 3D ha permesso il rilevamento della nascente localizzazione dell'RNA nei nuclei perché inizialmente è stato ottimizzato per esaminare l'espressione genica e gli stati di cromatina durante la differenziazione delle cellule germinali testicolari mediante ibridazione della fluorescenza dell'RNA in situ (FISH)2,3. Questo metodo 3D è applicabile anche alla combinazione di immunofluorescenza, DNA e RNA FISH. Inoltre, questo metodo ha aiutato nella scoperta della cromatina sessuale postmeiotica (PMSC), un compartimento silenzioso dei cromosomi sessuali trovato negli spermatidi postmeiotici2.
Il metodo di scorrimento 3D è stato originariamente ottimizzato attraverso la combinazione di due fasi essenziali di preparazione dello scivolo comunemente utilizzate per la colorazione nucleare: la fase di fissazione progettata per fissare i materiali nucleari e la fase di permeabilizzazione destinata a rimuovere i materiali citoplasmatici al fine di migliorare l'accessibilità dei reagenti di colorazione, come anticorpi e sonde FISH. Come descritto in precedenza inun'altra pubblicazione 3, è stato determinato che la fase di permeabilizzazione deve precedere la fase di fissazione al fine di ottenere risultati ottimali con basso background citoplasmatico. In questo metodo di scorrimento 3D, la fase di permeabilizzazione e la successiva fase di fissazione vengono eseguite direttamente su tubuli seminiferi e sono seguite dalla dissociazione meccanica delle cellule germinali con forcep prima della citospinning su diapositive. Un metodo alternativo per RNA FISH di cellule spermatogeniche è stato sviluppato in un altro laboratorio4. In questo metodo, coerentemente con il metodo 3D, la fase di permeabilizzazione deve precedere la fase di fissazione per ottenere risultati ottimali di RNA FISH.
Il seguente protocollo descrive il metodo di scorrimento 3D e fornisce un esempio di una possibile applicazione, Cot-1 RNA FISH per il rilevamento di RNA nascenti nucleari. Il DNA Cot-1 è costituito da elementi ripetitivi nel genoma. Qui, una sonda del DNA Cot-1 è ibridata ad un introne e a un UTR delle trascrizioni nascenti, rilevando così le regioni trascrittili attive nel nucleo5,6. Le diapositive 3D sono versatili e possono essere applicate a una combinazione di tecniche di immunofluorescenza, DNA e RNA FISH al fine di ottenere un esame dettagliato delle relazioni spaziali tra l'architettura della cromatina.

<table border="0" cellpadding="0" cellspacing="0" width="562">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Cot-1 DNA&#160;</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:119px;">18440-016</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stratagene Prime-It Fluor Fluorescence Labeling Kit</td>
			<td style="width:147px;">Agilent</td>
			<td align="right" style="width:119px;">
			300380
			</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Herring sperm DNA Solution</td>
			<td style="width:147px;">Invitrogen</td>
			<td>15634-017</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol, 200 proof (absolute)</td>
			<td>Pharmco-AAPER</td>
			<td align="right">111000200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium acetate</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">S2889</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Formamide deionized</td>
			<td>American Bioanalytical</td>
			<td>AB00600-00500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium citrate tribasic dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">C8532</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Bovine serum albumin</td>
			<td style="width:147px;">Lifeblood Medical</td>
			<td style="width:119px;">77110-100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextran sulfate sodium salt from Leuconostoc spp.</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">D6001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:119px;">A10491-01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">S0389</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">PIPES</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">P6757</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium chloride hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">M0250</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">T8787</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td align="right" style="width:119px;">76240</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td align="right" style="width:119px;">221465</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Phosphate buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">P4417</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VECTASHIELD Mounting Medium with DAPI</td>
			<td style="width:147px;">Vector Laboratories</td>
			<td style="width:119px;">H1200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ribonucleoside-vanadyl complex</td>
			<td>New England Biolabs</td>
			<td style="width:119px;">S1402S</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Illustra MicroSpin G-50 Columns</td>
			<td>GE Healthcare</td>
			<td style="width:119px;">27-5330-01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Microcentrifuge</td>
			<td style="width:147px;">Eppendorf</td>
			<td align="right" style="width:119px;">5424</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Forceps</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">300-050</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Microcentrifuge tubes</td>
			<td style="width:147px;">Bioexpress</td>
			<td style="width:119px;">C-3262-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Cytospin 3</td>
			<td style="width:147px;">Shandon</td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Coplin jar</td>
			<td style="width:147px;">Fisher</td>
			<td style="width:119px;">08-815</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superfrost /Plus Microscope Slides</td>
			<td>Fisher</td>
			<td>12-550-15</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-well dish</td>
			<td>Fisher</td>
			<td style="width:119px;">12-566-301</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cover glass&#160;</td>
			<td>Fisher</td>
			<td style="width:119px;">12-544-G</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">Air incubator</td>
			<td style="width:147px;">Revolutionary Science</td>
			<td style="width:119px;">RS-IF-203</td>
		</tr>
	</tbody>
</table>

slide preparation method to preserve three-dimensional chromatin architecture of testicular germ cells

Durante la differenziazione testicolare delle cellule germinali, la struttura della cromatina nucleare cambia dinamicamente. Di seguito viene descritto un metodo progettato per preservare la disposizione tridimensionale della cromatina delle cellule germinali testicolari presenti nei topi; questo metodo è stato definito come il metodo di scorrimento tridimensionale (3D). In questo metodo, i tubuli testicolari vengono trattati direttamente con una fase di permeabilizzazione che rimuove il materiale citoplasmatico, seguito da una fase di fissazione che fissa i materiali nucleari. I tubuli vengono quindi dissociati, la sospensione cellulare è citospun e le cellule aderiscono alle diapositive. Questo metodo migliora la sensibilità verso il rilevamento di strutture subnucleari ed è applicabile per l'immunofluorescenza, il DNA e la fluorescenza dell'RNA nell'ibridazione in situ (FISH) e la combinazione di questi metodi di rilevamento. Come esempio di una possibile applicazione del metodo di scorrimento 3D, viene mostrato un COT-1 RNA FISH per rilevare gli RNA nascenti. Il metodo di scorrimento 3D faciliterà l'esame dettagliato delle relazioni spaziali tra la struttura della cromatina, il DNA e l'RNA durante la differenziazione delle cellule germinali testicolari.

Un risultato rappresentativo di una diapositiva 3D è mostrato nella figura 2. In questo esperimento, Cot-1 RNA FISH è stato eseguito per rilevare la trascrizione nascente insieme all'immunostaining usando anticorpi anti CBX1 e γH2AX. Per combinare RNA FISH e immunostaining, l'immunostaining è stato eseguito per primo, seguito da RNA FISH come descritto in3. La CBX1 è una proteina eterocromatina che si localizza in eterocromatina pericentromerica e cromosomi sessuali silenziosi in meiosi c...

Watch this Scientific Journal Video about Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells at JoVE.com

Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells

Il materiale qui descrive un metodo sviluppato per preservare la struttura tridimensionale della cromatina delle cellule germinali testicolari. Questo è stato definito il metodo di scorrimento tridimensionale (3D). Questo metodo migliora la sensibilità per il rilevamento di strutture subnucleari ed è applicabile per l'immunofluorescenza, il DNA e la fluorescenza dell'RNA nell'ibridazione in situ (FISH).

Metodo di preparazione delle diapositive per preservare l'architettura tridimensionale della cromatina delle cellule germinali testicolari

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve ...

slide-preparation-method-to-preserve-three-dimensional-chromatin

Research

JoVE Journal

Biology

7.3K Views.  Cincinnati Children's Hospital Medical Center. Il materiale qui descrive un metodo sviluppato per preservare la struttura tridimensionale della cromatina delle cellule germinali testicolari. Questo è stato definito il metodo di scorrimento tridimensionale (3D). Questo metodo migliora la sensibilità per il rilevamento di strutture subnucleari ed è applicabile per l'immunofluorescenza, il DNA e la fluorescenza dell'RNA nell'ibridazione in situ (FISH).

Video: Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

Microfabbricazione di chip di dimensioni Ponteggi per tridimensionale coltura cellulare

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

Ricerca

Biologia

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

Hi-C: un metodo per studiare l&#39;architettura tridimensionale dei genomi.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

Isolamento di cellule epiteliali mammarie da tridimensionale Mixed-cell Spheroid Co-cultura

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses1.
 Various strategies such as Staput2,3, centrifugal elutriation1, and flow cytometry (FC)4,5 have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies. 
It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates - which tend to form because of the syncytial nature of the seminiferous epithelium6,7 - and lack cell clumps1 . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones1 . On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types1,8 . 
	The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting4, and ulterior gene expression studies9 .

A novel protocol for the mechanical preparation of testicular cell suspensions from rodent material, avoiding enzymes and detergents, is described. The method is very simple, fast, reproducible, and renders good quality cell suspensions, which are suitable for flow sorting and RNA extraction.

Tecnica semplice ed efficace per la preparazione delle sospensioni cellulari testicolari

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

Un metodo efficiente per quantitativa, analisi singola cella della cromatina Cambiamento e architettura nucleare in tutto il montaggio Ovuli in<em&gt; Arabidopsis</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

FRET Imaging in idrogel tridimensionali

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine system, but the complex structural relationships and three-dimensional (3D) vasculature architectures of the ovary are not well described. In order to visualize the 3D connections and architecture of blood vessels in the intact ovary, the first important step is to make the ovary optically clear. In order to avoid tissue shrinkage, we used the hydrogel fixation-based passive CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/ Immunostaining/In situ-hybridization-compatible Tissue Hydrogel) protocol method to clear an intact ovary. Immunostaining, advanced multiphoton confocal microscopy, and 3D image-reconstructions were then used for the visualization of ovarian vessels and follicular capillaries. Using this approach, we showed a significant positive correlation (P &#60;0.01) between the length of the follicular capillaries and volume of the follicular wall.

Here we present an adaptation of the passive CLARITY and 3D reconstruction method for visualization of the ovarian vasculature and follicular capillaries in intact mouse ovaries.

Ricostruzione tridimensionale dell'architettura vascolare dell'ovaia passiva sdoganate per la chiarezza del Mouse

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo-Clear: Un tessuto metodo di compensazione per l'Imaging tridimensionale del tessuto adiposo

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

Risoluzione a una cellula di immagini tridimensionali di organoidi intatti

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...

Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular matrix. The ability of CSCs to participate in the development of endothelium constitutes an important characteristic that directly contributes to the general understanding of the mechanisms of tumorigenesis and tumor metastasis. The purpose of this work is to establish a reproducible methodology to investigate the tumor-initiation capability of ovarian cancer stem cells (OCSCs). Herein, we examined the neovascularization mechanism between endothelial cells and OCSCs along with the morphological changes of endothelial cells using the in vitro co-culture model NICO-1. This protocol allows visualization of the neovascularization step surrounding the OCSCs in a time course manner. The technique can provide insight regarding the angiogenetic properties of OCSCs in tumor metastasis.

Ovarian cancer stem cells (OCSC) are responsible for cancer initiation, recurrence, therapeutic resistance, and metastasis. The OCSC vascular niche is considered to promote self-renewal of OCSCs, leading to chemoresistance. This protocol provides the basis for establishing a reproducible OCSC vascular niche model in vitro.

Valutazione delle proprietà angiogenetiche delle cellule staminali del cancro ovarico utilizzando il sistema di co-coltura tridimensionale, NICO-1

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular ...