Questo lavoro è stato sostenuto dal Fonds Igiene dell &#39;Università Medica di Graz. Tutti i soggetti hanno dato il loro consenso informato. Approvazione istituzionale del protocollo di studio è stato ottenuto presso l&#39;Università Medica di Graz.

1. Prelievo dei campioni <ol><li> Rimuovere fisso palatale espansori dopo 4 mesi di servizio intraorale. Utilizzare Sistema Campo Asciutto NOLA per isolare gli apparecchi prima di togliere loro (figure 1 e 2). </li><li> Utilizzare una pinza sterilizzata, guanti e vassoi per rimuovere espansori senza l&#39;aggiunta di contaminazione (figure 3 e 4). </li><li> Oggetti Conservare in Sarstedt 120 fiale ml a -20 ° C. Prendere per laboratorio su ghiaccio e processo entro 24 ore. </li></ol> 2. Biofilm fissazione <ol><li> Raschiare fiocchi biofilm con un bisturi sterile, o raccogliere i pezzi con una pinza sterile. </li><li> Aggiungi ghiacciata soluzione al 4% PFA fino campione è ben coperto. </li><li> Incubare la miscela a +4 ° C (non congelare) per 3 a 12 ore. Tempi di fissazione più o temperature superiori fissazione può rendere le buste di cellule meno permeabile a sonde oligonucleotidiche gram-negativi. </li><li> Rimuovere la soluzione PFA e lavare con ghiacciata PBS 1X. Ripetere questa operazione 2-3 volte al rTogliete residuo PFA. </li><li> Risospendere il campione in 1 vol. ghiacciato 1X PBS e aggiungere 1 vol. ghiacciato del 96% (v / v) di etanolo. </li><li> Conservare il campione a -20 ° C. Campioni fissati in base a questo protocollo può essere conservato per diversi mesi o anni. </li></ol> 3. Disidratazione dei campioni fisso <ol><li> Applicare 5-30 microlitri di PFA materiale fissato campione di un vetrino da microscopio. </li><li> Secco a 46 ° C per circa 15 minuti oa temperatura ambiente per più tempo. Scongelare il lisozima e la formammide. </li><li> Aggiungere 250 ml di lisozima (1mg/ml) a temperatura ambiente per 10 minuti, facilitando la penetrazione delle sonde nelle cellule dalla distruzione parziale delle pareti cellulari. </li><li> Dip scivolare nel 50%, 80% e 96% (v / v) di etanolo per 3 minuti ciascuno. L&#39;effetto disidratante della serie concentrazione di etanolo si disintegrerà delle membrane cellulari. </li><li> Asciugare i vetrini a 46 ° C per 10 min. </li></ol> 4. Ibridazione in situ <ol><li> Preparare 1 ml diibridazione tampone fresco (vedi Tabella 1 per le concentrazioni). Concentrazioni formammide utilizzato in questo studio: 10% (EUBmix), 20% (Bac303, dei conti correnti postali, MUT590) o 45% (LGCmix). </li><li> Scongelare le soluzioni oligonucleotide sonda. Sonde scongelati devono essere tenuti sul ghiaccio e al riparo dalla luce. </li><li> Aggiungere 2 ml di ciascuna sonda a 200 l di tampone di ibridazione, mescolare bene e applicare la miscela al campione disidratato su un vetrino da microscopio. </li><li> Mettere un pezzo di carta velina in un piatto piazza Petri e versare il buffer di ibridazione rimanente sulla carta velina. </li><li> Collocare immediatamente la diapositiva orizzontalmente nel piatto e chiudere il piatto. Incubare in un forno a 46 ° C per 1-5 ore (90 min sarà sufficiente nella maggior parte dei casi). Le funzioni piatto come una camera di umidità impedendo l&#39;evaporazione della soluzione di ibridazione dalla diapositiva. In particolare, l&#39;evaporazione di formammide può causare non sonda specifica vincolante per non colpire le cellule. </li><li> Preparare 50 ml di tampone di lavaggio (vedi Tabella 2 per concentrations). Preparare il tampone di lavaggio in un tubo da 50 ml e preriscaldare a 48 ° C a bagnomaria. La fase di lavaggio viene eseguita a 48 ° C. </li><li> Rimuovere il piatto con la diapositiva dal forno di ibridazione. Immediatamente lavare il buffer di ibridazione con un piccolo volume di pre-riscaldato tampone di lavaggio, e trasferire la diapositiva nel buffer di lavaggio rimanenti. </li><li> Posizionare il tubo contenente il tampone di lavaggio e lo scivolo di nuovo nel bagno d&#39;acqua e incubare a 48 ° C per 10-15 minuti. </li><li> Rimuove la pagina dal tubo e tuffo nella gelida DDH 2 O per 2-3 secondi per eliminare residui di tampone di lavaggio. </li><li> Asciugare la diapositiva il più rapidamente possibile (l&#39;uso di aria compressa è raccomandato). Essiccazione rapida riduce sonda dissociazione. </li><li> Diapositive secchi possono essere conservati al buio a -20 ° C per diverse settimane senza significative perdite di sonda-conferito segnale di fluorescenza. </li></ol> 5. Microscopia <ol><li> Dopo la FISH e lavaggio, apply due gocce di antifadent vicino alle estremità destra e sinistra di una diapositiva (fette congelato dovrebbe essere portata a temperatura ambiente prima di questo passo). </li><li> Mettere un coperchio microscopio scivolare sulla parte superiore e attendere che il antifadent si è diffuso oltre l&#39;intera diapositiva. Si noti che una quantità eccessiva di antifadent può sfocare l&#39;immagine microscopio. </li><li> Osservare i campioni al microscopio confocale a scansione laser dotati di filtri adatti o laser. Abbiamo usato una Leica TCS unità (HCX PL APO/63x; NA 1.2). I dati possono essere analizzati con software come IMARIS o AMIRA. </li><li> Diapositive integrati nel antifadent possono essere conservati a +4 ° C (non congelare) per un massimo di 7 giorni prima la sonda-conferito fluorescenza comincia a declinare. In alternativa, il antifadent può essere rimosso con DDH 2 O, e le diapositive secca può essere conservata a -20 ° C per un periodo prolungato di tempo. </li></ol> 6. Rappresentante dei risultati: Raschiando biofilm off apparecchi ortodontici fissi (Figura 5) produce fiocchi adatto (Figura 6) che può essere ibridato direttamente su vetrini rivestiti per microscopia. In questo modo, i diversi gruppi di batteri orobiome possono essere identificati nel loro naturale ambiente tridimensionale da rRNA tagging batterica etichettati in modo diverso sonde specifiche (figure 7 e 8). Nella Figura 7, biofilm era macchiata di EUBmix (verde, tutti i batteri) e LGCmix (giallo, Firmicutes). Firmicutes visualizzati in verde, in quanto sono state colorate di giallo e blu. Nella Figura 8, biofilm era macchiata di EUBmix (rosso, tutti i batteri), Bac303 (blu, Bacteroidetes) e dei conti correnti postali (giallo, Porphyromonas gingivalis). Porphyromonas gingivalis è mostrata in bianco giallognolo, in quanto tutte e tre le sonde si legano al suo DNA e la sovrapposizione dei risultati di colori in un bianco segnale. Differenze morfologiche tra i gruppi di batteri possono essere identificati (Figure 9 e 10). Grandi gruppi di batteri coccoidi sono mostrati in figura 9, dove è stata eseguita con EUBmix colorazione (verde, tutti i bacteria). Diverse forme di batteri orali sono stati visualizzati in Figura 10, dove i batteri filamentosi coccoidi e possono essere distinte dalla colorazione con EUBmix (rosso). Inoltre, un tipico fungo-come la struttura del biofilm possono essere trattati tramite modellazione 3D dei dati CLSM (figure 11 e 12) <a href="http://www.jove.com/files/ftp_upload/2967/Film1 Klug Santigli 01_2_JES_Web.mov">Clicca qui per guardare un film della modellazione 3D.</a> <img alt="Figura 1" src="/files/ftp_upload/2967/2967fig1.jpg" /> Figura 1. Espansore palatale fisso in situ. <img alt="Figura 2" src="/files/ftp_upload/2967/2967fig2.jpg" /> Figura 2. Sistema campo Nola a secco. <img alt="Figura 3" src="/files/ftp_upload/2967/2967fig3.jpg" /> Figura 3. Pinze sterili, guanti e vassoio. <img alt="Figura 4" src="/files/ftp_upload/2967/2967fig4.jpg" /> Figura 4. Rimosso espansione. <img alt="Figura 5" src="/files/ftp_upload/2967/2967fig5.jpg" /> Figura 5. Biofilm raschiando con un bisturi sterile. <img alt="Figura 6" src="/files/ftp_upload/2967/2967fig6.jpg" /> Fiocchi di resina Figura 6. Ibridato direttamente su lastre di vetro rivestito. <img alt="Figura 7" src="/files/ftp_upload/2967/2967fig7.jpg" /> Figura 7 immagine CLSM:. Differenziazione di uno specifico gruppo di batteri. Sovrapposizione di dati 2D CLSM pila 3D. Biofilm colorati con EUBmix (verde, tutti i batteri) e LGCmix (giallo, Firmicutes). <img alt="Figura 8" src="/files/ftp_upload/2967/2967fig8.jpg" /> Figura 8 immagine CLSM:. Differenziazione di uno specifico gruppo di batteri. Sovrapposizione di dati 2D CLSM pila 3D. Macchiato biofilm con EUBmix (rosso, tutti i batteri), Bac303 (blu, Bacteroidetes) e dei conti correnti postali (giallo, Porphyromonas gingivalis). <img alt="Figura 9" src="/files/ftp_upload/2967/2967fig9.jpg" /> Figura 9 immagine CLSM:. Differenziazione delle morfologie specifiche. Sovrapposizione di dati 2D CLSM pila 3D. Biofilm colorati con EUBmix (verde, tutti i batteri). Grandi cluster di batteri coccoidi (frecce). <img alt="Figura 10" src="/files/ftp_upload/2967/2967fig10.jpg" /> . Figura 10 immagini CLSM: differenziazione delle morfologie specifiche. Sovrapposizione di dati 2D CLSM pila 3D. Biofilm colorati con EUBmix (rosso, tutti i batteri). Coccoidi (freccia in basso) e batteri filamentosi (freccia in alto) si possono distinguere. <img alt="Figura 11" src="/files/ftp_upload/2967/2967fig11.jpg" /> Figura 11. Struttura di funghi, viste 3D dal basso. Pile di fiocchi biofilm (raschiato la superficie di un apparecchio ortodontico), colorate con EUBmix e proceduressed con IMARIS (immagine CLSM). <img alt="Figura 11" src="/files/ftp_upload/2967/2967fig11.jpg" /> Figura 12. Struttura di funghi, vista laterale 3D. Pile di fiocchi biofilm (raschiato la superficie di un apparecchio ortodontico) trattati con IMARIS (immagine CLSM). <table border="1"><tbody><tr><td colspan="4"> Ibridazione di buffer (200 mL) </td></tr><tr><td> Concentrazione Formammide </td><td> 10% </td><td> 20% </td><td> 45% </td></tr><tr><td> 5 M NaCl </td><td> 36 </td><td> 36 </td><td> 36 </td></tr><tr><td> 1 M Tris-HCl </td><td> 4 </td><td> 4 </td><td> 4 </td></tr><tr><td> 2% SDS </td><td> 1 </td><td> 1 </td><td> 1 </td></tr><tr><td> FA </td><td> 20 </td><td> 40 </td><td> 90 </td></tr><tr><td><strong&gt; DDH 2 O </td><td> 138 </td><td> 118 </td><td> 68 </td></tr><tr><td> Ogni pesce sonda </td><td> 2 </td><td> 2 </td><td> 2 </td></tr></tbody></table> Costituenti Tabella 1. Tampone di ibridazione (concentrazioni in microlitri). <table border="1"><tbody><tr><td colspan="4"> Tampone di lavaggio (50 ml) </td></tr><tr><td> Concentrazione Formammide </td><td> 10% </td><td> 20% </td><td> 45% </td></tr><tr><td> 5 M NaCl </td><td> 4500 </td><td> 2150 </td><td> 300 </td></tr><tr><td> 1 M Tris-HCl </td><td> 1000 </td><td> 1000 </td><td> 1000 </td></tr><tr><td> 0,5 M EDTA </td><td> 0 </td><td> 500 </td><td> 500 </td></tr><tr><td> DDH 2 O </td><td> 44 500 </td><td> 46 350 </td&gt; <td> 48 200 </td></tr></tbody></table> Costituenti Tabella 2. Tampone di lavaggio (concentrazioni in microlitri). Movie 1. <a href="http://www.jove.com/files/ftp_upload/2967/Film1 Klug Santigli 01_2_JES_Web.mov">Clicca qui per guardare il film.</a>

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Nessun conflitto di interessi dichiarati.

Il presente protocollo descritto è un approccio altamente funzionale per biofilm colorazione raccolti dai materiali duri. Campionamento e ibridazione sono i passaggi più critici. Durante il campionamento, occorre fare attenzione che le fette di spessore adeguato sono raccolti per assicurare che la struttura del biofilm rimane intatto. Durante l&#39;ibridazione, è essenziale per evitare eccessive fluttuazioni di temperatura, evitando legame non specifico, o la perdita di legame, delle sonde fluorescente. Questo protocollo FISH è un metodo elegante per macchiare normale biofilm eterogeneo direttamente su vetrini senza interrompere la sua composizione. Le slide possono essere immediatamente utilizzati per la microscopia, senza la necessità di spostare il campione ancora una volta. In questo modo, un miglioramento rispetto colorazione in tubi si ottiene una minore forza di taglio applicata al biofilm. Fette&gt; 50 micron di spessore possono essere preparati e studiati in questo modo.

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

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

Microscopia confocale a scansione laser (CLSM) di naturale biofilm eterogeneo è oggi facilitato da una gamma completa di tecniche di colorazione, uno dei quali è ibridazione in situ fluorescente (FISH). 1,2 Abbiamo eseguito uno studio pilota in cui campioni di biofilm orali raccolte da fisso apparecchi ortodontici (palatale expander) sono state macchiate dalla FISH, con l&#39;obiettivo di valutare l&#39;tridimensionale organizzazione del biofilm naturale e accumulo di placca. 3,4 PESCE crea l&#39;opportunità di macchia cellule nel loro ambiente nativo biofilm con l&#39;uso di 16S fluorescente rRNA-targeting sonde. 4-7,19 Rispetto alle tecniche alternative come l&#39;etichettatura immunofluorescenza, questo è un poco costoso, tecnica di etichettatura preciso e semplice da indagare diversi gruppi di batteri nel biofilm consorzi misti. 18,20 sonde sono state usate Generale che si legano al Eubatteri ( EUB338 + + EUB338II EUB338III, di seguito EUBmix), 8-10 Firmicutes (LGC354 AC;. LGCmix di seguito), 9,10 e Bacteroidetes (Bac303) 11 Inoltre, le sonde specifiche vincolanti di Streptococcus mutans (MUT590) 12,13 e Porphyromonas gingivalis (conti correnti postali) 13,14 sono stati utilizzati . La durezza estrema dei materiali superficie interessata (in acciaio inox e resina acrilica) ci costrinse a trovare nuovi modi di preparare il biofilm. Poiché questi materiali di superficie non potrebbe essere facilmente tagliato con una cryotome, vari metodi di campionamento sono stati esplorati per ottenere intatto biofilm orale. La più realizzabile di questi approcci è presentata in questa comunicazione. Piccoli fiocchi del biofilm che trasporta resina acrilica sono stati raschiata con un bisturi sterile, facendo attenzione a non danneggiare la struttura del biofilm. Forcipe sono stati utilizzati per raccogliere biofilm dalle superfici in acciaio. Una volta raccolti, i campioni sono stati fissati e posti direttamente sul polysine vetrini rivestiti. FISH è stata eseguita direttamente su queste diapositive con le sonde mentioned sopra. PESCE protocolli diversi sono stati combinati e modificati per creare un nuovo protocollo che è stato facile da maneggiare. 5,10,14,15 Successivamente i campioni sono stati analizzati al microscopio confocale a scansione laser. Noto configurazioni 3,4,16,17 potrebbero essere visualizzati, tra cui funghi in stile formazioni e gruppi di batteri coccoidi pervaso da canali. Inoltre, la composizione batterica di queste strutture tipiche biofilm sono stati analizzati e immagini 2D e 3D creato.

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

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

Vi presentiamo un protocollo per l&#39;analisi strutturale e compositiva delle risorse naturali biofilm orale da apparecchi ortodontici con<em&gt; In situ</em&gt; Ibridazione (FISH) e la microscopia confocale a scansione laser (CLSM). Campioni di biofilm orale sono stati raccolti da espansori palatali, raschiando acrilica sfalda la loro superficie e riferimento per l&#39;elaborazione molecolare.

Analisi biofilm orale di espansori palatali da Fluorescence In Situ-ibridazione e microscopia confocale a scansione laser

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

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

Research

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Medicine

15.9K Views.  Medical University of Graz. Vi presentiamo un protocollo per l'analisi strutturale e compositiva delle risorse naturali biofilm orale da apparecchi ortodontici con In situ Ibridazione (FISH) e la microscopia confocale a scansione laser (CLSM). Campioni di biofilm orale sono stati raccolti da espansori palatali, raschiando acrilica sfalda la loro superficie e riferimento per l'elaborazione molecolare.

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

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

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

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

Probe-based Endomicroscopy laser confocale del tratto urinario: La Tecnica

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

Ricerca

Medicina

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

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

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

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

Analisi clinicopatologica di miRNA espressione in Breast Cancer tessuti mediante l&#39;utilizzo di miRNA<em&gt; In Situ</em&gt; ibridazione

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

Oral Biofilm Sampling for Microbiome Analysis in Healthy Children

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

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

Biofilm orali campionamento per l'analisi di Microbiome nei bambini sani

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

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

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

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

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

Seconda generazione di armoniche segnali coniglio sclera come strumento per la valutazione del tessuto terapeutico reticolazione (TXL) per la miopia

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

Oral Biofilm Formation on Different Materials for Dental Implants

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

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

Formazione di Biofilm orali su differenti materiali per impianti dentali

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

Oral Health Assessment by Lay Personnel for Older Adults

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

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

Valutazione della salute orale da parte del personale laico per gli adulti più anziani

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

Visualizing Membrane Ruffle Formation using Scanning Electron Microscopy

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

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

Visualizzazione della formazione di increspature a membrana utilizzando la microscopia elettronica a scansione

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

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

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

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

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

Ibridazione in situ a fluorescenza interfase di strisci di midollo osseo di mieloma multiplo

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

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

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

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

Quantificazione planimetrica semi-automatica della placca dentale mediante una telecamera a fluorescenza intraorale

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

Acquisizione di campioni di tessuto orale di fibrosi sottomucosa per l'analisi spazio-omica

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

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

Autore in primo piano: Svelare i misteri delle neoplasie maligne orali 

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

Isolamento di cellule con informazioni morfologiche e spaziali da campioni di fibrosi sottomucosa orale mediante microdissezione a cattura laser

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