We would like to acknowledge our colleague Dr. Jalaiah Varikooty who provided insight and expertise on the topic of the lid wiper, that greatly assisted this research.

Ethik-Anweisung: Vor der Zellen aus menschlichen Patienten zu sammeln, informierte Zustimmung und Ethik Genehmigung eingeholt werden muss. 1. Bereiten Sie Stain HINWEIS: Bereiten Sie Fleck am Tag des Experiments. <ol><li> Werden 5 ul Ethidium (oder 4 &amp; mgr; M) und 5 &amp; mgr; l Calcein AM (oder 4 &amp; mgr; M) zu 2,5 ml Phosphat gepufferter Salzlösung (PBS) in 15 ml Zentrifugenröhrchen; mischen. Wickeln Sie in Aluminiumfolie vor Licht und lagern bei RT bis zur Verwendung zu schützen. </li></ol> 2. Sammeln Zellen <ol><li> Entfernen Zellkultureinsätze aus ihrer Verpackung und Beschriftung für jede Augenlid abgetastet werden. Mark Orientierung der Zellsammlung auf der Seite der Membran Kunststoffhalter mit Permanentmarker. Conduct Spaltlampe Inspektion bei mäßiger Vergrößerung (etwa 20fach) eine angemessene Gesundheits der Region zu bestätigen IC zu unterziehen. </li><li> Einen Tropfen Lokalanästhetikum (0,5% Proparacainhydrochlorid) im unteren Bindehautsack von each Auge und weisen Teilnehmer Augen zu halten für eine Minute geschlossen. </li><li> Evert oberen Deckel Auge und halten Sie ihn durch die Wimpern; Kontakt mit dem Deckel Wischbereich zu vermeiden. Optional: die Hilfe eines sekundären Ermittler kann diesen Schritt auszuführen, erforderlich. </li><li> Anwenden Membran senkrecht auf dem Mittelteil des Deckels Wischbereich mit minimalem Druck. </li><li> Halten Membran anstelle 3 - 5 sec. Beachten Sie die Membran durchscheinend im Kontaktbereich. An diesem Punkt, entfernen Sie die Membran und ermöglichen dem Teilnehmer Deckel zu &quot;kippen&quot; zurück an ihren Platz. </li><li> Unverzüglich einem Tropfen PBS auf die Probe anzuwenden, um es zu verhindern das Austrocknen. Membran schaltet lässig und sollte nicht drehen opaque jederzeit, da dies Trocknen angibt. Haben die Sekundär Ermittler gehen sofort mit der Bearbeitung der Proben (Abschnitt 3). </li><li> Führen Prüfung endgültig Auge Integrität der Bindehaut zu gewährleisten. Dispense Augen Schmierstoffe und anweisen Teilnehmerreiben sich die Augen zu vermeiden, bis betäubende Wirkung nachlässt (ca. 15 min.). </li></ol> 3. Probenverarbeitung <ol><li> Mit Mikro-Schere, schneiden Sie die Membran in die Hälfte (die eine Hälfte für jede der Downstream-Analysen), die senkrecht durch die Mitte der Zellsammlung. Schneiden Sie entlang der äußeren Rand einer Membran Hälfte aus dem Kunststoffhalter zu trennen; vermeiden Wechselwirkung mit den gesammelten Zellen auf der Membran, auch sichergestellt wird, dass die Membran nicht auf sich selbst falten wird. </li><li> Sichern Sie die Membran Pinzette vor dem vollständigen Lösen von dem Halter verwenden. Platz Membran in markierte 2 ml Zentrifugenröhrchen mit 500 ul 95% (v / v) Ethanol und lassen Sie für mindestens 20 Minuten bis maximal 2 Stunden zu beheben, bevor die Verarbeitung (Abschnitt 4.2). </li><li> Unmittelbar separate zweite Hälfte der Membran (wie in Abschnitt 3.2 beschrieben) und prompt mit Abschnitt 4.1 fortfahren. </li></ol> 4. Probenfärbungs <ol><li> Immunocytochemical Anfärben <ol><li> Vorsichtig Membran in 35 mm Glasboden Kulturschale mit Sammlung Seite nach unten; gewährleisten Membran ist flach. </li><li> Je 20 ul immunzytochemischer Zusammensetzung Fleck versammelt in Abschnitt 1 auf der Membran. Wenn Probe Locken, verwenden Sie Einweg-Pipettenspitzen vorsichtig die Membran flach an den Kanten nach unten drücken. Vermeiden Sie den Kontakt mit der Zellsammelgebieten. </li><li> Sanft decken die Membran mit Deckglas. Vermeiden Sie unnötige Bewegung der Probe. Deckel Schüssel mit Deckel und Dichtung mit Labor-Film an den Rändern. Nicht Transparenz der Schale (oben und unten) und die Sichtbarkeit der Probe behindern. Beschriften mit Labor-Marker. </li><li> Unmittelbar gehen mit Bildgebung (Abschnitt 5.1), dann wieder auf Abschnitt 4.2 mit zytologische Färbung der anderen Hälfte der Membran fortzusetzen. </li></ol></li><li> Die zytologische Färbung <ol><li> Allmählich Membran Hydrat langsam 500 ul destilliertem Wasser zu dem Rohr Zugabe used zur Fixierung in Abschnitt 3.4. Absaugen Inhalt mehrmals in Pipettenspitze zu mischen, dann Inhalt zu entfernen. </li><li> In 500 ul destilliertem Wasser. stehen lassen für ein paar Sekunden, dann entfernen. In 500 ul alcianblau und lassen für 3 min. Entfernen Fleck und führen Sie 3 aufeinanderfolgende 500 &amp; mgr; l destilliertes Wasser spült oder bis die Flüssigkeit spült klar. </li><li> In 500 ul Hematoxylin # 1 Fleck und lassen für 3 min. Entfernen Fleck und führen in drei aufeinander folgenden 500 &amp; mgr; l destilliertes Wasser Spülungen oder bis die Flüssigkeit spült klar. Entwässern (Rückseite der Abschnitt 4.2.1) durch die klare Flüssigkeit abgesaugt. </li><li> In 500 ul Papanicolaou OG-6 Fleck und lassen für 3 min. Entfernen Fleck und führen Sie einen 500 &amp; mgr; l 95% (v / v) Ethanol spülen. </li><li> In 500 ul Papanicolaou EA-65 Fleck und lassen für 3 min. Entfernen stain und führen 3 aufeinanderfolgende 500 &amp; mgr; l 95% (v / v) Ethanol Spülungen. </li><li> Fully dehydratisieren drei aufeinanderfolgende 500 &amp; mgr; l 100% Ethanol Spülungen verwendet. Mit TWEezers, entfernen Probe aus der Lösung; vermeiden Sie das Berühren Zellsammelgebiete. </li><li> Legen Membran auf Glasobjektträger; diese Zelle Sammelleitung sicherzustellen, ist entweder parallel oder senkrecht Rand zur leichteren Ausrichtung während der Bildgebung zu gleiten. </li><li> Einen Tropfen von 100% Ethanol und Deckel mit Glas rutschen. Unmittelbar gehen mit Bildgebung (Abschnitt 5.2). </li></ol></li></ol> 5. Beispiel Imaging <ol><li> Imaging Fluorescent Immunzytochemische Stains <ol><li> Verwenden eines konfokalen Laserscanning - Mikroskops (CLSM) 11 oder Fluoreszenzmikroskop 12, mit einer digitalen Farbkamera ausgestattet ist , mit einem Computer verbunden Software eine Bildaufnahme ausgeführt wird . Aufgrund der Proben innerhalb Kulturschalen enthalten ist, kann ein umgekehrtes Mikroskop erforderlich sein. </li><li> Set up Mikroskop 11,12 nach Absorptions- und Emissionsspektren von Ethidium Homodimer-1 (528/617 nm Ex / Em maxima in Gegenwart von DNA) und Calcein AM (494/517 nm Ex / Em maxima). </li><li> Wählen Sie die kleinste Vergrößerung des Mikroskops (zB 2.5X Objektiv) und aktivieren Sie den Digital - Imaging - System; beachten Sie die auf dem Computermonitor angezeigt Probe. </li><li> Überprüfen Sie Probe für zelluläre Funktionen von Interesse. </li><li> Wählen Sie die gewünschte Mikroskopvergrößerung und Bilder aufzunehmen, Imaging-Software. </li><li> Speichern von Dateien in entweder nativ Mikroskop - Dateiformat oder ein unkomprimiertes Dateiformat (zB * .raw, * .tif). </li></ol></li><li> Imaging zytologische Stains <ol><li> Mit einem Standard-Laborhellfeldmikroskop ohne Filter, mit einer digitalen Farbkamera ausgestattet ist, mit einem Computer verbunden Software eine Bildaufnahme ausgeführt wird. </li><li> Platz und Schloss Glasträger (aus Schritt 4.2.8) auf Mikroskoptisch. Rehydrieren Probe mit 100% Ethanol je nach Bedarf über den gesamten Abbildungsverfahren. </li><li> Wählen Sie die kleinste Vergrößerung des Mikroskops (zB 2.5X Objektiv) und aktivieren the Digital-Imaging-System; beachten Sie die auf dem Computermonitor angezeigt Probe. </li><li> Stellen Sie sicher, Zellsammlung ist entweder mit X oder Y-Achse des Mikroskoptisch Steuerung ausgerichtet. Stellen Sie, wenn nötig, durch das Deckglas zu entfernen und durch Drehen der Probe mit einer Pipettenspitze oder mit einer Pinzette. </li><li> Unter Verwendung der Imaging-Software, Capture-Bild (er) der gesamten Probe mit dem niedrigsten Mikroskopvergrößerung. </li><li> Wechseln Sie zu moderaten Mikroskopvergrößerung (zB 10 - 20fach - Objektiv) und justieren Mikroskop Lichtquelle und Software Abbildungsparameter (Belichtung, Kontrast, Weißabgleich, etc.), und deaktivieren Sie die automatische Software Metering. Speichern Sie diese Einstellungen für die zukünftige Verwendung. </li><li> Bestimmen Sie Anfangs- und Endpunkte von Scan (zB oben links und unten rechts Ecken der Zellsammlung). </li><li> Unter Verwendung der Imaging-Software, Capture-Bild (er) der gesamten Zelle Sammelbereich innerhalb der Anfangs- und Endpunkte. Stellen Sie sicher, 20% von Seite zu Seite und von oben nach unten überlappen between alle benachbarten Bildern. </li><li> Speichern von Dateien mit dem nativen Mikroskop - Dateiformat oder ein unkomprimiertes Dateiformat (zB * .raw, * .tif). </li><li> Generieren endgültige Panoramabild mit automatisierten Stitching - Software der Wahl (zB Adobe Photoshop Elements). Verwenden Sie Referenzbilder im Schritt genommen 5.2.5), um die Genauigkeit der automatischen Heften im endgültigen Bild zu bestätigen. </li></ol></li></ol>

<ol>
	<li>Barbato, G., 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=Diurnal+variation+in+spontaneous+eye-blink+rate.">Diurnal variation in spontaneous eye-blink rate.</a> Psychiatry Res. 93, 145-151 (2000).</li><li>Korb, D. R., 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=Lid-Wiper+Epitheliopathy+and+Dry-Eye+Symptoms+in+Contact+Lens+Wearers.">Lid-Wiper Epitheliopathy and Dry-Eye Symptoms in Contact Lens Wearers.</a> Eye &amp; Contact Lens. 28, 211-216 (2002).</li><li>Dumbleton, K., Woods, C. A., Jones, L. W., Fonn, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+contemporary+contact+lenses+on+contact+lens+discontinuation.">The impact of contemporary contact lenses on contact lens discontinuation.</a> Eye &amp; Contact Lens. 39, 93-99 (2013).</li><li>Roba, M., Duncan, E., Hill, G., Spencer, N., Tosatti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Friction+measurements+on+contact+lenses+in+their+operating+environment.">Friction measurements on contact lenses in their operating environment.</a> Tribol Lett. 44, 387-397 (2011).</li><li>Sickenberger, W., Pult, H., Sickenberger, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LIPCOF+and+contact+lens+wearers:+a+new+tool+to+forecast+subjective+dryness+and+degree+of+comfort+of+contact+lens+wearers.">LIPCOF and contact lens wearers: a new tool to forecast subjective dryness and degree of comfort of contact lens wearers.</a> Contactologia. 22, 74-79 (2000).</li><li>Pult, H., Purslow, C., Berry, M., Murphy, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+tests+for+successful+contact+lens+wear:+relationship+and+predictive+potential.">Clinical tests for successful contact lens wear: relationship and predictive potential.</a> Optom Vis Sci. 85, E924-E929 (2008).</li><li>Doughty, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+features+of+cells+along+Marx's+line+of+the+marginal+conjunctiva+of+the+human+eyelid.">Morphological features of cells along Marx's line of the marginal conjunctiva of the human eyelid.</a> Clin Exp Optom. 96, 76-84 (2013).</li><li>Jalbert, I., 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=Assessing+the+human+lid+margin+epithelium+using+impression+cytology.">Assessing the human lid margin epithelium using impression cytology.</a> Acta Ophthalm. 90, e547-e552 (2012).</li><li>Nelson, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impression+cytology.">Impression cytology.</a> Cornea. 7, 71-81 (1988).</li><li>Egbert, P. R., Lauber, S., Maurice, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+conjunctival+biopsy.">A simple conjunctival biopsy.</a> Am J Ophthalmol. 84, 798-801 (1977).</li><li>Wilson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Confocal microscopy. , 1-64 (1990).</li><li>Denk, W., Strickler, J. H., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+laser+scanning+fluorescence+microscopy.">Two-photon laser scanning fluorescence microscopy.</a> Science. 248, 73-76 (1990).</li><li>Tomlins, P., Roy, P., Curnow, J., Rauz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+the+EyePRIM+Device+for+Conjunctival+Impression+for+Flow+Cytometry.">Assessment of the EyePRIM Device for Conjunctival Impression for Flow Cytometry.</a> Invest Ophthalm Vis Sci. 54, 5430-5430 (2013).</li><li>Knop, E., Korb, D. R., Blackie, C. A., Knop, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lid+margin+is+an+underestimated+structure+for+preservation+of+ocular+surface+health+and+development+of+dry+eye+disease.">The lid margin is an underestimated structure for preservation of ocular surface health and development of dry eye disease.</a> Res Proj Dry Eye Syn. 45, 108-122 (2010).</li><li>Knop, E., 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=The+lid+wiper+and+muco-cutaneous+junction+anatomy+of+the+human+eyelid+margins:+an+in+vivo+confocal+and+histological+study.">The lid wiper and muco-cutaneous junction anatomy of the human eyelid margins: an in vivo confocal and histological study.</a> J Anat. 218, 449-461 (2011).</li><li>Christensen, J. A., Skaarland, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+and+cell+area+measurements+in+the+cytological+evaluation+of+pleural+effusions:+a+study+of+subjective+assessments+and+morphometric+measurements.">Nuclear and cell area measurements in the cytological evaluation of pleural effusions: a study of subjective assessments and morphometric measurements.</a> Diag Cytopathol. 3, 50-54 (1987).</li></ol>

The authors have no relevant funding or conflicts to disclose.

Impressionszytologie wird typischerweise auf der bulbären Bindehaut durchgeführt, gemischte Celluloseestermembranen. Proben werden aus Schüttgut Blätter zugeschnitten, sterilisiert, aufgetragen und von der Oberfläche entfernt konjunktivale mit einer Pinzette. Unter Verwendung der gleichen Membranmaterial, ein kolbengesteuertes wurde IC - Vorrichtung vor kurzem entwickelt , um die Oberflächengeometrie der bulbären Bindehaut zu entsprechen, und aufrechtzuerhalten konsistente Druck zwischen Anwendungen 13.</sup...

Der menschliche Oberlid führt rund 10.000 blinkt täglich 1, mit nur 1 - 2 mm schmaler konjunktivaler Region die Augapfels zu widersetzen . Aufgrund seiner Wischbewegung während des Blink, dieser Teil der Lidrand hat den &quot;Deckel Wischer&quot; Bereich 2 bezeichnet worden. Es wird angenommen, dass die Reibung in diesem Bereich während der gewöhnlichen Blinzeln erhöht wird, aufgrund der Deckel über der Kugel Abwischen. Dies kann eine wichtige Rolle in der Augen Komfort spielen. Kontaktlinsenträger insbesondere Erfahrung Augenbeschwerden und dies ist einer der Hauptgründe für Aufhören Linse 3 tragen. Wenn eine Kontaktlinse auf dem Auge platziert ist, ist der Reibungskoeffizient zwischen dem Linsenmaterial und der Augenoberfläche sich gezeigt , 4 zu ändern. Es gibt Hinweise darauf , dass Trockenheit Symptome auf diese veränderte Reibung 2,5,6 zusammenhängen könnte. Dieser Verein kann auch in klinisch beobachtbaren Phänomenen reflektiert werden, insbesondere Deckel wipro Epitheliopathie (LWE), auch oberen Lidrand - Färbung (ULMS) 6 genannt. LWE ist ein frühes Zeichen der Augenreizung und ein potentieller Prädiktor für trockene Augenkrankheit. Es wird beobachtet, und durch die Vitalfärbung der oberen und unteren Deckelrandbereichen gemessen. Während dieses Grading - System 2 und ihrer klinischen Gültigkeit (dh Korrelation mit subjektiven Symptomen) noch immer umstritten sind, bleibt Augenbeschwerden ein Rätsel für Kliniker und Wissenschaftler gleichermaßen und, was am wichtigsten ist , eine Unannehmlichkeit für den Patienten. Bis heute ist wenig über klinisch relevanten Veränderungen in der (Teil-) Zelluläre Anatomie und Physiologie des Bereichs Deckel Wischer bekannt 7 und lediglich ein Bericht macht Gebrauch von zytologischen Methoden 8. Impression Zytologie (IC) ist seit mehr als 40 Jahren beschäftigt Zellen aus der epithelialen Oberfläche des Bulbus oder Tarsalbindehaut durch Anwendung einer 9,10 Membran zu sammeln. Nach dem Entfernen erfahren die anhaftenden Zellen cytological Färbung und mikroskopische Bildgebung. Aufgrund der anatomischen und zellulären Unterschiede im Deckel Wischbereich erfordert diese Technik Optimierung.

<table border="0" cellpadding="0" cellspacing="0" width="753">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:349px;">Alcian Blue, 1% in 3% Acetic Acid</td>
			<td style="width:119px;">Sigma</td>
			<td style="width:119px;">B8438-250ML</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hematoxylin, Gill 1</td>
			<td style="width:119px;">Sigma</td>
			<td>GHS116-500ML</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Papanicolaou stain, OG-6</td>
			<td style="width:119px;">Sigma</td>
			<td>HT40116-500 ml</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Papanicolaou stain, Modified EA</td>
			<td style="width:119px;">Sigma</td>
			<td>HT40232-1L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Live/Dead Viability/Cytotoxicity Kit</td>
			<td>Life Technologies</td>
			<td>L-3224</td>
			<td>contains ethidium homodimer-1 and Calcein AM dyes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alcaine (0.5% proparacaine hydrochloride)</td>
			<td>Alcon</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Millicell Cell Culture Insert, 12&#160;mm, hydrophilic PTFE, 0.4&#160;&#181;m</td>
			<td>EMD Millipore</td>
			<td>PICM01250&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">35 mm Glass Bottom Dishes</td>
			<td>MatTek Corporation</td>
			<td>P35G-0-20-C</td>
			<td></td>
		</tr>
	</tbody>
</table>

impression cytology of the lid wiper area

Few reports on the cellular anatomy of the lid wiper (LW) area of the inner eyelid exist and only one report makes use of cytological methods. The optimization of a method of collecting, staining and imaging cells from the LW region using impression cytology (IC) is described in this study. Cells are collected from the inner surface of the upper eyelid of human subjects using hydrophilic polytetrafluoroethylene (PTFE) membranes, and stained with cytological dyes to reveal the presence of goblet cells, mucins, cell nuclei and various degrees of pre- and para-keratinization. Immunocytochemical dyes show cell esterase activity and compromised cell membranes by the use of a confocal scanning laser microscope. Up to 100 microscopic digital images are captured for each sample and stitched into a high-resolution, large scale image of the entire IC span. We demonstrate a higher sensitivity of IC than reported before, appropriate for identifying cellular morphologies and metabolic activity in the LW area. To our knowledge, this is the first time this selection of fluorescent dyes was used to image LW IC membranes. This protocol will be effective in future studies to reveal undocumented details of the LW area, such as assessing cellular particularities of contact lens wearers or patients with dry eye or lid wiper epitheliopathy.

Die Zellkulturmembran über den Kunststoffhalter aufgespannt ist ein praktisches Werkzeug für Handheld-Sammlung von Epithelzellen aus dem Deckel Wischer Konjunktiven. Dieses Verfahren eliminiert die Notwendigkeit für zusätzliche sterilisierten Instrumente typischerweise verwendet zur Vorbereitung und IC Membranen verarbeiten. 1 ist die IC des Deckels Wischbereich stellt einen Zellkultureinsatz verwendet wird . Wir optimiert zwei verschiedenen Färbungsprotokolle, die ...

Watch this Scientific Journal Video about Impression Cytology of the Lid Wiper Area at JoVE.com

Impression Cytology of the Lid Wiper Area

We collected, stained and imaged cells from the conjunctiva of the inner eyelid margin of human subjects. By characterizing cell morphology and metabolic activity, this method may further our understanding of dry eye and the role that friction between the ocular surfaces may play in perceiving ocular discomfort.

Impressionszytologie des Deckels Wiper Bereich

Few reports on the cellular anatomy of the lid wiper (LW) area of the inner eyelid exist and only one report makes use of cytological methods. The ...

impression-cytology-of-the-lid-wiper-area

Research

JoVE Journal

Medicine

10.3K Aufrufe.  University of Waterloo. We collected, stained and imaged cells from the conjunctiva of the inner eyelid margin of human subjects. By characterizing cell morphology and metabolic activity, this method may further our understanding of dry eye and the role that friction between the ocular surfaces may play in perceiving ocular discomfort. 

Serie: Impression Cytology of the Lid Wiper Area

Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of hospitalizations in children with a public health impact similar to that of Rotavirus. NoVs are RNA viruses of great genetic diversity and there is a continuous appearance of new strains. Five genogroups are recognized; GI and GII with their many genotypes and subtypes being the most important for human infection. However, the diagnosis of these two genotypes remains problematic, delaying diagnosis and treatment. 1, 2, 3
For RNA extraction from stool specimens the most commonly used method is the QIAmp Viral RNA commercial kit from Qiagen. This method combines the binding properties of a silica gel membrane, buffers that control RNases and provide optimum binding of the RNA to the column together with the speed of microspin. This method is simple, fast and reliable and is carried out in a few steps that are detailed in the description provided by the manufacturer. 
 Norovirus is second only to rotavirus as the most common cause of diarrhea. Norovirus diagnosis should be available in all studies on pathogenesis of diarrhea as well as in outbreaks or individual diarrhea cases. At present however norovirus diagnosis is restricted to only a few centers due to the lack of simple methods of diagnosis. This delays diagnosis and treatment 1, 2, 3. In addition, due to costs and regulated transportation of corrosive buffers within and between countries use of these manufactured kits poses logistical problems. As a result, in this protocol we describe an alternative, economic, in-house method which is based on the original Boom et al. method4 which uses the nucleic acid binding properties of silica particles together with the anti-nuclease properties of guanidinium thiocyanate.
For the detection and genogrouping (GI and GII) of NoVs isolates from stool specimens, several RT-PCR protocols utilizing different targets have been developed. The consensus is that an RT-PCR using TaqMan chemistry would be the best molecular technique for diagnosis, because it combines high sensitivity, specificity and reproducibility with high throughput and ease of use. Here we describe an assay targeting the open reading frame 1 (ORF1)-ORF2 junction region; the most conserved region of the NoV genome and hence most suitable for diagnosis. For further genetic analysis a conventional RT-PCR that targets the highly variable N-terminal-shell from the major protein of the capsid (Region C) using primers originally described by Kojima et al. 5 is detailed. Sequencing of the PCR product from the conventional PCR enables the differentiation of genotypes belonging to the GI and GII genogroups.

A One-Step RT-PCR assay for detection and genogroup identification of Norovirus isolates from children&#x2019;s stools, that utilizes primers and TaqMan probes specific to the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the Norovirus genome is described. A non-commercial, cost-effective RNA extraction method is detailed.

Erkennung und Genogrouping von Noroviren aus Kinder-Hocker mit der TaqMan One-Step RT-PCR

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of ...

Forschung

Medizin

Immunohistochemical Staining of B7-H1 (PD-L1) on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue

B7-H1/PD-L1, a member of the B7 family of immune-regulatory cell-surface proteins, plays an important role in the negative regulation of cell-mediated immune responses through its interaction with its receptor, programmed death-1 (PD-1) 1,2. Overexpression of B7-H1 by tumor cells has been noted in a number of human cancers, including melanoma, glioblastoma, and carcinomas of the lung, breast, colon, ovary, and renal cells, and has been shown to impair anti-tumor T-cell immunity3-8.
Recently, B7-H1 expression by pancreatic adenocarcinoma tissues has been identified as a potential prognostic marker9,10. Additionally, blockade of B7-H1 in a mouse model of pancreatic cancer has been shown to produce an anti-tumor response11. These data suggest the importance of B7-H1 as a potential therapeutic target. Anti-B7-H1 blockade antibodies are therefore being tested in clinical trials for multiple human solid tumors including melanoma and cancers of lung, colon, kidney, stomach and pancreas12.
In order to eventually be able to identify the patients who will benefit from B7-H1 targeting therapies, it is critical to investigate the correlation between expression and localization of B7-H1 and patient response to treatment with B7-H1 blockade antibodies. Examining the expression of B7-H1 in human pancreatic adenocarcinoma tissues through immunohistochemistry will give a better understanding of how this co-inhibitory signaling molecule contributes to the suppression of antitumor immunity in the tumor's microenvironment. The anti-B7-H1 monoclonal antibody (clone 5H1) developed by Chen and coworkers has been shown to produce reliable staining results in cryosections of multiple types of human neoplastic tissues4,8, but staining on paraffin-embedded slides had been a challenge until recently13-18. We have developed the B7-H1 staining protocol for paraffin-embedded slides of pancreatic adenocarcinoma tissues. The B7-H1 staining protocol described here produces consistent membranous and cytoplasmic staining of B7-H1 with little background.

Immunohistochemical Staining of B7-H1 PD-L1 on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue

B7-H1 (PD-L1) and its binding to PD-1 provide a major tumor-induced immunosuppressive signal in the tumor&#x2019;s microenvironment. An immunohistochemical staining technique to characterize the expression and localization of B7-H1 in pancreatic adenocarcinoma is described here.

Immunhistochemische Färbung von B7-H1 (PD-L1) auf Paraffin-embedded Slides der Pankreaskarzinom Tissue

B7-H1/PD-L1, a member  of the B7 family of immune-regulatory cell-surface proteins, plays an important  role in the negative regulation of cell-mediated ...

NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited for studies of cardiac metabolism, which require the heart to perform work within the context of physiologic preload and afterload pressures. Neely introduced modifications to the Langendorff technique to establish appropriate left ventricular (LV) preload and afterload pressures3. The model is known as the isolated LV working heart model and has been used extensively to study LV performance and metabolism4-6. This model, however, does not provide a properly loaded right ventricle (RV). Demmy et al. first reported a biventricular model as a modification of the LV working heart model7, 8. They found that stroke volume, cardiac output, and pressure development improved in hearts converted from working LV mode to biventricular working mode8. A properly loaded RV also diminishes abnormal pressure gradients across the septum to improve septal function. Biventricular working hearts have been shown to maintain aortic output, pulmonary flow, mean aortic pressure, heart rate, and myocardial ATP levels for up to 3 hours8.
When studying the metabolic effects of myocardial injury, such as ischemia, it is often necessary to identify the location of the affected tissue. This can be done by imaging the fluorescence of NADH (the reduced form of nicotinamide adenine dinucleotide)9-11, a coenzyme found in large quantities in the mitochondria. NADH fluorescence (fNADH) displays a near linearly inverse relationship with local oxygen concentration12 and provides a measure of mitochondrial redox state13. fNADH imaging during hypoxic and ischemic conditions has been used as a dye-free method to identify hypoxic regions14, 15 and to monitor the progression of hypoxic conditions over time10.
The objective of the method is to monitor the mitochondrial redox state of biventricular working hearts during protocols that alter the rate of myocyte metabolism or induce hypoxia or create a combination of the two. Hearts from New Zealand white rabbits were connected to a biventricular working heart system (Hugo Sachs Elektronik) and perfused with modified Krebs-Henseleit solution16 at 37 &deg;C. Aortic, LV, pulmonary artery, and left &amp; right atrial pressures were recorded. Electrical activity was measured using a monophasic action potential electrode. To image fNADH, light from a mercury lamp was filtered (350&plusmn;25 nm) and used to illuminate the epicardium. Emitted light was filtered (460&plusmn;20 nm) and imaged using a CCD camera. Changes in the epicardial fNADH of biventricular working hearts during different pacing rates are presented. The combination of the heart model and fNADH imaging provides a new and valuable experimental tool for studying acute cardiac pathologies within the context of realistic physiological conditions.

The objective is to monitor the mitochondrial redox state of isolated hearts within the context of physiologic preload and afterload pressures. A biventricular working rabbit heart model is presented. High spatiotemporal resolution fluorescence imaging of NADH is used to monitor the mitochondrial redox state of epicardial tissue.

NADH Fluoreszenz-Imaging von isolierten biventrikulären Arbeiten Kaninchenherzen

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited ...

Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, particularly in low dosage values1&#8203;&#8203;. &#8216;How to use PSA&#39; remains a current issue, either for diagnosis as a gray zone corresponding to a concentration in serum of 2.5-10 ng/ml which does not allow a clear differentiation to be made between cancer and noncancer2 or for patient follow-up as analysis of post-operative PSA kinetic parameters can pose considerable challenges for their practical application3,4. Alternatively, noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease, e.g. PCA3 in prostate cancer5,6 and to reveal uncharacterized aspects of tumor biology. Moreover, data from the ENCODE project published in 2012 showed that different RNA types cover about 62% of the genome. It also appears that the amount of transcriptional regulatory motifs is at least 4.5x higher than the one corresponding to protein-coding exons. Thus, long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) constitute a wide range of putative/candidate transcriptional regulatory sequences, as it is their primary function in infectious retroviruses. HERVs, which are spread throughout the human genome, originate from ancestral and independent infections within the germ line, followed by copy-paste propagation processes and leading to multicopy families occupying 8% of the human genome (note that exons span 2% of our genome). Some HERV loci still express proteins that have been associated with several pathologies including cancer7-10. We have designed a high-density microarray, in Affymetrix format, aiming to optimally characterize individual HERV loci expression, in order to better understand whether they can be active, if they drive ncRNA transcription or modulate coding gene expression. This tool has been applied in the prostate cancer field (Figure 1).

Human endogenous retroviruses (HERV), which occupy 8% of the human genome, retain scarce coding capacities but a hundred thousand long terminal repeats (LTRs). A custom Affymetrix microarray was designed to identify individual HERV locus expression and was used on prostate cancer tissues as a proof of concept for future clinical studies.

Microarray-basierte Identifizierung von Einzel HERV Expression Loci: Anwendung auf Biomarker Discovery in Prostatakrebs

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

Sammlung und Extraktion von DNA-Speichel für Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

Ein &quot;Patient-Like&quot; Orthotope Syngene Maus-Modell der Leberzellkarzinom Metastasen

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

Induction of Ischemic Stroke and Ischemia-reperfusion in Mice Using the Middle Artery Occlusion Technique and Visualization of Infarct Area

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of stroke on tissue injury and to evaluate the effectiveness of therapeutic interventions, several experimental models in a variety of species were developed. They include complete global cerebral ischemia, incomplete global ischemia, focal cerebral ischemia, and multifocal cerebral ischemia. The model described in this protocol is based on the middle cerebral artery occlusion (MCAO) and is related to the focal ischemia category. This technique produces consistent focal ischemia in a strictly defined region of the hemisphere and is less invasive than other methods. The procedure described is performed on mice, given the availability of several genetic variants and the high number of tests standardized for mice to aid in the behavioral and neurodeficit evaluation.

We describe a mouse model of stroke induced by the occlusion of the middle cerebral artery using a silicone coated suture. The protocol can be applied to induce permanent occlusion or a temporary ischemia, followed by reperfusion.

Die Induktion von ischämischem Schlaganfall und Ischämie-Reperfusion bei Mäusen die Mittel Artery Occlusion Technik und Visualisierung von Infarktbereich verwenden

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of ...

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This compound is currently under investigation as a PET tracer for demyelination. We recently described a novel chemical reaction to produce metafluorinated pyridines consisting of direct fluorination of a pyridine N-oxide and the utilization of this reaction for the radiochemical synthesis of [18F]3F4AP. In this article, we demonstrate how to produce this tracer using an automated synthesizer and an in-house made flow hydrogenation reactor. We also show the standard quality control procedures performed before releasing the radiotracer for preclinical animal imaging studies. This semi-automated procedure may serve as the basis for future production of [18F]3F4AP for clinical studies.

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

We demonstrate the semi-automated radiochemical synthesis of [18F]3F4AP and quality control procedures.

Automatisierte radiochemische Synthese von [<sup&gt; 18</sup&gt; F] 3F4AP: Ein neuartiger PET-Tracer zur Bildgebung von demyelinisierenden Krankheiten

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This ...

Tubal Cytology of the Fallopian Tube as a Promising Tool for Ovarian Cancer Early Detection

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to the lack of markers or tools for the ovarian cancer identification, the early detection of HGSC remains challenging. Direct sampling of the fallopian tube can enhance sensitivity for detection of neoplastic cells when the tumor is not grossly visible. We developed a procedure to collect fallopian tube cells directly from freshly received surgical specimens, which has shown excellent correlation with histological findings. This approach lays a foundation for the future utility of minimally invasive laparoscopic screening in high-risk patient populations.

We explored a tubal cytologic method by sampling the fallopian tube directly post-surgical excision as a tool of ovarian cancer early detection. Here, we present a protocol to collect fallopian tube cells from freshly received surgical specimens.

Tubal-Zytologie der Eileiter als vielversprechendes Werkzeug für Eierstockkrebs Früherkennung

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to ...

Accessing the Cytotoxicity and Cell Response to Biomaterials

Biomaterials contact directly or indirectly with the human tissues, making it important to evaluate its cytotoxicity. This evaluation can be performed by several methods, but a high discrepancy exists between the approaches used, compromising the reproducibility and the comparison among the obtained results. In this paper, we propose a protocol to evaluate biomaterials cytotoxicity using soluble extracts, which we use for dental biomaterials. The extracts preparation is detailed, from pellets production to its extraction in a culture medium. The biomaterials cytotoxicity evaluation is based on metabolic activity using the MTT assay, cell viability using the Sulphorhodamine B (SBR) assay, cell death profile by flow cytometry, and cell morphology using May-Gr&#252;nwald Giemsa. Additional to cytotoxicity evaluation, a protocol to evaluate cell function is described based on the expression of specific markers assessed by immunocytochemistry and PCR. This protocol provides a comprehensive guide for biomaterials cytotoxicity and cellular effects evaluation, using the extracts methodology, in a reproducible and robust manner.

This methodology aims to evaluate biomaterial cytotoxicity through the preparation of soluble extracts, using viability assays and phenotypic analysis, including flow cytometry, RT-PCR, immunocytochemistry, and other cellular and molecular biology techniques.

Zugang zur Zytotoxizität und Zellantwort auf Biomaterialien

Biomaterials contact directly or indirectly with the human tissues, making it important to evaluate its cytotoxicity. This evaluation can be performed by ...

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