Ce travail a été soutenu par le Fonds d&#39;hygiène de l&#39;Université médicale de Graz. Tous les sujets ont donné leur consentement éclairé. L&#39;approbation institutionnelle du protocole d&#39;étude a été obtenue de l&#39;Université médicale de Graz.

1. Prélèvement <ol><li> Retirer fixes palatine expandeurs après 4 mois de service intra-buccale. Utilisation du système NOLA terrain sec pour isoler les appareils avant leur suppression (figures 1 et 2). </li><li> Utilisez des pinces stérilisées, des gants et des plateaux pour enlever expandeurs sans ajouter la contamination (figures 3 et 4). </li><li> Objets magasin dans des flacons de 120 ml Sarstedt à -20 ° C. Prenez au laboratoire sur de la glace et le processus dans les 24 heures. </li></ol> 2. Fixation du biofilm <ol><li> Grattez flocons biofilm avec un scalpel stérile, ou de recueillir des pièces avec des pinces stériles. </li><li> Ajouter une solution glacée de 4% jusqu&#39;en PFA échantillon est bien couvert. </li><li> Incuber le mélange à 4 ° C (ne pas congeler) pour 3 à 12 heures. Temps de fixation plus longues ou à des températures supérieures de fixation peut rendre les enveloppes cellulaires des bactéries gram-négatives cellules moins perméable aux sondes oligonucléotidiques. </li><li> Retirer solution de PFA et laver avec glacée PBS 1X. Répétez cette étape 2-3 fois à reMove résiduelle PFA. </li><li> Resuspendre l&#39;échantillon en 1 vol. glacée PBS 1X et ajouter 1 vol. glacée 96% (v / v) d&#39;éthanol. </li><li> Conserver l&#39;échantillon à -20 ° C. Les échantillons fixés en fonction de ce protocole peuvent être stockés pendant plusieurs mois à plusieurs années. </li></ol> 3. La déshydratation des échantillons fixés <ol><li> Appliquer ul 50-30 du PFA matériel fixé l&#39;échantillon sur une lame de microscope. </li><li> Sec à 46 ° C pendant environ 15 min ou à température ambiante pendant plus longtemps. Décongeler le lysozyme et la formamide. </li><li> Ajouter 250 pi de lysozyme (1mg/ml) à température ambiante pendant 10 min, facilitant ainsi la pénétration des sondes dans les cellules par une destruction partielle des parois cellulaires. </li><li> Trempez glisser dans 50%, 80% et 96% (v / v) d&#39;éthanol pendant 3 min chacune. L&#39;effet diurétique de la série concentration d&#39;éthanol va se désintégrer les membranes cellulaires. </li><li> Sécher les lames à 46 ° C pendant 10 min. </li></ol> 4. Hybridation in situ <ol><li> Préparer 1 ml detampon d&#39;hybridation douce (voir le tableau 1 pour les concentrations). Le formamide concentrations utilisées dans cette étude: 10% (EUBmix), 20% (Bac303, POGI, MUT590) ou 45% (LGCmix). </li><li> Décongeler les solutions de sondes oligonucléotidiques. Sondes décongelés doivent être conservés sur la glace et protégé de la lumière. </li><li> Ajouter 2 ul de chaque sonde à 200 pi de tampon d&#39;hybridation, bien mélanger et appliquer le mélange à l&#39;échantillon déshydraté sur une lame de microscope. </li><li> Placez un morceau de papier de soie dans un plat carré de Pétri et versez le tampon d&#39;hybridation reste sur le papier de soie. </li><li> Immédiatement placer la lame à l&#39;horizontale dans le plat et fermer le plat. Incuber dans un four à 46 ° C pendant 1-5 heures (90 min suffiront dans la plupart des cas). Les fonctions plat comme une chambre de l&#39;humidité empêchant l&#39;évaporation de la solution d&#39;hybridation de la diapositive. En particulier, l&#39;évaporation de formamide peut provoquer non spécifiques se liant à la sonde cellules non cibles. </li><li> Préparer 50 ml de tampon de lavage (voir Tableau 2 pour concentrations). Préparer le tampon de lavage dans un tube de 50 ml et préchauffer à 48 ° C dans un bain d&#39;eau. L&#39;étape de lavage est effectué à 48 ° C. </li><li> Retirer le plat avec la lame du four d&#39;hybridation. Laver immédiatement le tampon d&#39;hybridation avec un petit volume de tampon de lavage pré-chauffé, et le transfert de la glisser dans le tampon de lavage restant. </li><li> Placer le tube contenant le tampon de lavage et de la glisser dans le bain d&#39;eau et incuber à 48 ° C pendant 10-15 min. </li><li> Retirer glisser dans le tube et plonger dans la glace froide O ddH 2 pour 2-3 secondes pour éliminer les résidus de tampon de lavage. </li><li> Sécher à l&#39;air de la diapositive aussi rapidement que possible (l&#39;utilisation d&#39;air comprimé est recommandé). Séchage rapide permettra de réduire la sonde de dissociation. </li><li> Diapositives séchées peuvent être stockés dans l&#39;obscurité à -20 ° C pendant plusieurs semaines sans perte significative de sonde conféré signal de fluorescence. </li></ol> 5. Microscopie <ol><li> Après FISH et le lavage, apply deux gouttes de près antifadent les extrémités gauche et droite d&#39;une diapositive (tranches congelé doit être réchauffé à la température ambiante avant cette étape). </li><li> Placez un couvercle sur le dessus de glissement de microscope et attendre que le antifadent s&#39;est répandu sur la lame entière. Notez qu&#39;une quantité excessive de antifadent peuvent brouiller l&#39;image du microscope. </li><li> Observez les échantillons sous un microscope confocal à balayage laser équipé de filtres appropriés ou des lasers. Nous avons utilisé un Leica TCS unité (HCX PL APO/63x; NA 1.2). Les données peuvent être analysées avec un logiciel tel que Imaris ou Amira. </li><li> Diapositives intégrées dans antifadent peuvent être conservés à 4 ° C (ne pas congeler) jusqu&#39;à 7 jours avant la sonde de fluorescence conféré commence à diminuer. Alternativement, le antifadent peuvent être enlevés avec ddH 2 O, et les lames séchées peuvent être conservées à -20 ° C pour une période de temps prolongée. </li></ol> 6. Les résultats représentatifs: Grattage biofilm hors appareils orthodontiques fixes (Figure 5) de flocons de rendements adéquats (figure 6) qui peuvent être hybridées directement sur lames de verre recouvertes de microscopie. De cette façon, les différents groupes de bactéries orobiome peuvent être identifiés dans leur milieu naturel en trois dimensions environnement en marquage ARNr bactériens étiquetés différemment des sondes spécifiques (figures 7 et 8). Dans la figure 7, le biofilm était tachée de EUBmix (vert, toutes les bactéries) et LGCmix (jaune, Firmicutes). Firmicutes apparaissent en vert, comme ils ont été colorés avec du jaune et du bleu. Dans la figure 8, le biofilm était tachée de EUBmix (rouge, toutes les bactéries), Bac303 (bleu, Bacteroidetes) et POGI (jaune, Porphyromonas gingivalis). Porphyromonas gingivalis est montré dans blanc jaunâtre, comme tous les trois sondes se lient à l&#39;ADN et le chevauchement des résultats de couleurs dans un signal blanc. Les différences morphologiques entre les groupes de bactéries peuvent également être identifiés (figures 9 et 10). Grands groupes de bactéries coccoïdes sont présentés dans la figure 9, où la coloration a été réalisée avec EUBmix (vert, tous les bacteria). Différentes formes de bactéries orales ont été visualisées dans la figure 10, où les bactéries coccoïdes et filamenteuses peuvent être distingués par coloration avec EUBmix (rouge). En outre, un type de champignon comme la structure du biofilm peut être traitée via la modélisation 3D des données CLSM (figures 11 et 12) <a href="http://www.jove.com/files/ftp_upload/2967/Film1 Klug Santigli 01_2_JES_Web.mov">Cliquez ici pour regarder un film de la modélisation 3D.</a> <img alt="Figure 1" src="/files/ftp_upload/2967/2967fig1.jpg" /> Figure 1. Correction d&#39;extension palatine in situ. <img alt="Figure 2" src="/files/ftp_upload/2967/2967fig2.jpg" /> Figure 2. Système de Nola terrain sec. <img alt="Figure 3" src="/files/ftp_upload/2967/2967fig3.jpg" /> Figure 3. Stérilisé pinces, des gants et un plateau. <img alt="Figure 4" src="/files/ftp_upload/2967/2967fig4.jpg" /> Figure 4. Suppression d&#39;extension. <img alt="Figure 5" src="/files/ftp_upload/2967/2967fig5.jpg" /> Figure 5. Grattage biofilm avec un scalpel stérile. <img alt="Figure 6" src="/files/ftp_upload/2967/2967fig6.jpg" /> Flocons de résine figure 6. Directement hybridés sur des lames de verre enduit. <img alt="Figure 7" src="/files/ftp_upload/2967/2967fig7.jpg" /> Figure 7 Image CLSM:. Différenciation d&#39;un groupe spécifique de bactéries. Superposition de données 2D 3D pile CLSM. Biofilm colorés avec EUBmix (vert, toutes les bactéries) et LGCmix (jaune, Firmicutes). <img alt="Figure 8" src="/files/ftp_upload/2967/2967fig8.jpg" /> Figure 8 images CLSM:. Différenciation d&#39;un groupe spécifique de bactéries. Superposition de données 2D 3D pile CLSM. Tachés biofilm avec EUBmix (rouge, toutes les bactéries), Bac303 (bleu, Bacteroidetes) et POGI (jaune, Porphyromonas gingivalis). <img alt="Figure 9" src="/files/ftp_upload/2967/2967fig9.jpg" /> Figure 9 images CLSM:. Différenciation des morphologies particulières. Superposition de données 2D 3D pile CLSM. Biofilm colorés avec EUBmix (vert, toutes les bactéries). Grands groupes de bactéries coccoïdes (flèches). <img alt="Figure 10" src="/files/ftp_upload/2967/2967fig10.jpg" /> Images CLSM Figure 10:. Différenciation des morphologies particulières. Superposition de données 2D 3D pile CLSM. Biofilm colorés avec EUBmix (rouge, toutes les bactéries). Coccoïde (flèche bas) et des bactéries filamenteuses (flèche haut) peuvent être distingués. <img alt="Figure 11" src="/files/ftp_upload/2967/2967fig11.jpg" /> Structure de champignons Figure 11., Vues 3D à partir de ci-dessous. Piles de flocons de biofilm (gratté la surface d&#39;un appareil orthodontique), colorées avec EUBmix et procéduresSSED avec Imaris (image CLSM). <img alt="Figure 11" src="/files/ftp_upload/2967/2967fig11.jpg" /> Structure de champignons Figure 12., Vue de côté 3D. Piles de flocons de biofilm (gratté la surface d&#39;un appareil orthodontique) traitées avec Imaris (image CLSM). <table border="1"><tbody><tr><td colspan="4"> Tampon d&#39;hybridation (200 pi) </td></tr><tr><td> Concentration de formamide </td><td> 10% </td><td> 20% </td><td> 45% </td></tr><tr><td> 5 M de NaCl </td><td> 36 </td><td> 36 </td><td> 36 </td></tr><tr><td> 1 M Tris-HCl </td><td> 4 </td><td> 4 </td><td> 4 </td></tr><tr><td> 2% de SDS </td><td> 1 </td><td> 1 </td><td> 1 </td></tr><tr><td> FA </td><td> 20 </td><td> 40 </td><td> 90 </td></tr><tr><td><strong&gt; DdH 2 O </td><td> 138 </td><td> 118 </td><td> 68 </td></tr><tr><td> Toute sonde FISH </td><td> 2 </td><td> 2 </td><td> 2 </td></tr></tbody></table> Constituants Tableau 1. De tampon d&#39;hybridation (concentrations en pl). <table border="1"><tbody><tr><td colspan="4"> Tampon de lavage (50 ml) </td></tr><tr><td> Concentration de formamide </td><td> 10% </td><td> 20% </td><td> 45% </td></tr><tr><td> 5 M de NaCl </td><td> 4500 </td><td> 2150 </td><td> 300 </td></tr><tr><td> 1 M Tris-HCl </td><td> 1000 </td><td> 1000 </td><td> 1000 </td></tr><tr><td> EDTA 0,5 M </td><td> 0 </td><td> 500 </td><td> 500 </td></tr><tr><td> ddH 2 O </td><td> 44 500 </td><td> 46 350 </td&gt; <td> 48 200 </td></tr></tbody></table> Constituants Tableau 2. De tampon de lavage (concentrations en pl). Vidéo 1. <a href="http://www.jove.com/files/ftp_upload/2967/Film1 Klug Santigli 01_2_JES_Web.mov">Cliquez ici pour regarder le film.</a>

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Pas de conflits d&#39;intérêt déclarés.

Le présent protocole décrit une approche très réaliste de biofilms coloration recueillies à partir de matériaux durs. L&#39;échantillonnage et l&#39;hybridation sont les étapes les plus critiques. Lors de l&#39;échantillonnage, il faut prendre soin que les tranches d&#39;épaisseur suffisante, sont collectées afin de s&#39;assurer que la structure du biofilm reste intacte. Pendant l&#39;hybridation, il est essentiel d&#39;éviter les fluctuations excessives de température, évitant ainsi la liaison non spécifique, ou de perte de liaison, des sondes marquées par fluorescence. Ce protocole FISH est une méthode élégante pour colorer normale biofilm hétérogènes directement sur les lames de verre, sans perturber sa composition. Les diapositives peuvent être immédiatement utilisés pour la microscopie, sans besoin de déplacer l&#39;échantillon une fois de plus. De cette façon, une amélioration par rapport à coloration dans des tubes est réalisé par la force de cisaillement moins d&#39;être appliquée à la biofilm. Tranches&gt; 50 um d&#39;épaisseur peut être préparé et étudié dans ce mode.

<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

Microscopie confocale à balayage laser (CLSM) du biofilm hétérogènes naturel est aujourd&#39;hui facilitée par une gamme complète de techniques de coloration, l&#39;un d&#39;eux étant l&#39;hybridation fluorescente in situ (FISH) 1,2. Nous avons réalisé une étude pilote dans lequel des échantillons de biofilms oraux recueillis auprès fixes appareils orthodontiques (palatine expandeurs) ont été colorées par FISH, l&#39;objectif étant d&#39;évaluer l&#39;organisation tridimensionnelle des biofilms naturels et l&#39;accumulation de plaque. 3,4 POISSONS crée une opportunité pour colorer des cellules dans leur environnement natif du biofilm par l&#39;utilisation de 16S marqués par fluorescence ARNr de ciblage des sondes. 4-7,19 Comparé à d&#39;autres techniques comme l&#39;étiquetage d&#39;immunofluorescence, c&#39;est un peu coûteuse, la technique de l&#39;étiquetage précis et simple d&#39;enquêter sur différents groupes bactériens dans des consortiums mixtes biofilm. 18,20 sondes ont été utilisées général qui se lient aux eubactéries ( EUB338 + + EUB338II EUB338III, ci-après EUBmix), 8-10 Firmicutes (LGC354 AC;. LGCmix ci), 9,10 et Bacteroidetes (Bac303) 11 De plus, les sondes de liaison spécifique à Streptococcus mutans (MUT590), 12,13 et Porphyromonas gingivalis (POGI) 13,14 ont été utilisés . L&#39;extrême dureté des matériaux de surface impliquées (en acier inoxydable et résine acrylique) nous a obligés à trouver de nouvelles façons de préparer le biofilm. Comme ces matériaux de surface ne pouvait pas être facilement coupé avec un cryotome, diverses méthodes d&#39;échantillonnage ont été explorées afin d&#39;obtenir intacte biofilm orale. Le plus pratique de ces approches est présenté dans cette communication. De petits éclats de la résine acrylique biofilm porteurs ont été grattées avec un scalpel stérile, en prenant soin de ne pas endommager la structure du biofilm. Pince ont été utilisés pour recueillir des biofilms sur les surfaces en acier. Une fois collectés, les échantillons ont été fixés et placés directement sur polysine lames de verre recouvertes. FISH a été effectuée directement sur ces diapositives avec les sondes mentionnées ci-dessus. Protocoles divers poissons ont été combinés et modifiés pour créer un nouveau protocole qui a été facile à manipuler. 5,10,14,15 Par la suite, les échantillons ont été analysés par microscopie confocale à balayage laser. Bien connu des configurations 3,4,16,17 pourrait être visualisées, y compris les champignons de style formations et des grappes de bactéries coccoïdes envahi par des canaux. En outre, la composition bactérienne de ces structures typiques du biofilm ont été analysés et des images 2D et 3D créés.

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

Nous présentons un protocole pour l&#39;analyse structurelle et compositionnelle du biofilm naturel d&#39;administration orale à partir d&#39;appareils orthodontiques<em&gt; In situ</emL&#39;hybridation&gt; (FISH) et microscopie confocale à balayage laser (CLSM). Échantillons de biofilms oraux ont été recueillies auprès de expandeurs palatine, le grattage de résine acrylique s&#39;écaille leur surface et leur référence pour le traitement moléculaire.

Analyse orale de biofilm palatine Expandeurs par fluorescence hybridation in situ et la microscopie confocale à balayage 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

JoVE Journal

Medicine

15.9K vues.  Medical University of Graz. Nous présentons un protocole pour l'analyse structurelle et compositionnelle du biofilm naturel d'administration orale à partir d'appareils orthodontiques In situ

Vidéo: 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.

Basée sur sonde endomicroscopie confocale laser de l&#39;appareil urinaire: La Technique

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

Recherche

Médecine

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.

Analyse clinicopathologique de miRNA expression dans le cancer du sein à l&#39;aide de tissus miRNA<em&gt; In Situ</em&gt; hybridation

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.

Échantillonnage de Biofilm oral Microbiome analyse chez les enfants en bonne santé

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.

Deuxième génération d’harmoniques signaux dans la sclérotique de lapin comme un outil pour l’évaluation des thérapeutiques tissulaires réticulation (TXL) pour la myopie

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.

Formation de Biofilm oral sur différents matériaux pour Implants dentaires

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.

Évaluation de la santé buccodentaire par le personnel laïc pour les personnes âgées

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.

Visualisation de la formation de volants membranaires à l’aide de la microscopie électronique à balayage

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.

Fluorescence interphasique hybridation in situ des frottis de moelle osseuse du myélome multiple

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.

Quantification planimétrique semi-automatisée de la plaque dentaire à l’aide d’une caméra à fluorescence intra-orale

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

Capture d’échantillons de tissus de fibrose sous-muqueuse buccale pour l’analyse spatio-omique

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.

Pleins feux sur l’auteur : Percer les mystères des tumeurs malignes potentielles buccales 

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.

Isolement de cellules avec des informations morphologiques et spatiales à partir d’échantillons de fibrose sous-muqueuse buccale par microdissection par capture 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 ...