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.

Etica dichiarazione: prima di raccogliere le cellule da soggetti umani, consenso informato e l&#39;approvazione etica deve essere ottenuto. 1. Preparare Stain NOTA: Preparare macchia il giorno dell&#39;esperimento. <ol><li> Aggiungere 5 ml di etidio (o 4 micron) e 5 ml calceina AM (o 4 micron) a 2,5 ml di tampone fosfato salino (PBS) in 15 ml provetta da centrifuga; mescolare. Avvolgere in un foglio di alluminio per proteggere dalla luce e conservare a temperatura ambiente fino al momento dell&#39;uso. </li></ol> 2. Le cellule Raccogliere <ol><li> Rimuovere gli inserti colture cellulari dalla loro confezione e un&#39;etichetta per ogni palpebra da campionare. Mark orientamento della raccolta delle cellule sul lato del supporto in plastica a membrana usando pennarello indelebile. Comportamento fessura ispezione lampada con ingrandimento moderata (circa 20X) per confermare appropriata la salute della regione a subire IC. </li><li> Erogare una goccia di anestetico topico (0,5% proparacaina cloridrato) nel sacco congiuntivale inferiore each occhio e istruire partecipante di tenere gli occhi chiusi per un minuto. </li><li> Evert superiore palpebra e tenere in posizione dalle ciglia; evitare il contatto con l&#39;area coperchio tergicristallo. Optional: l&#39;aiuto di un investigatore secondaria può essere richiesto di eseguire questo passaggio. </li><li> Applicare membrana perpendicolarmente sulla parte centrale della regione tergicristallo coperchio con pressione minima. </li><li> Tenere membrana in posizione per 3-5 secondi. Osservare la membrana diventare trasparente nella zona di contatto. A questo punto, rimuovere delicatamente la membrana e permettere il coperchio del partecipante a &quot;flip&quot; al suo posto. </li><li> applicare prontamente una goccia di PBS al campione, per evitare che si secchi. Membrane diventerà traslucida e non deve girare opaco in qualsiasi momento, come indica essiccazione. Avere l&#39;investigatore secondaria a procedere immediatamente con l&#39;elaborazione di campioni (sezione 3). </li><li> Condurre controllo occhio finale per garantire l&#39;integrità della congiuntiva. Dispensare lubrificanti oculari e istruire partecipanteper evitare di strofinare gli occhi fino a effetto anestetico svanisce (circa 15 min.). </li></ol> Processing 3. Campione <ol><li> Utilizzando micro-forbici, tagliare la membrana a metà (la metà per ciascuna delle analisi a valle), perpendicolarmente attraverso il centro della raccolta delle cellule. Tagliare lungo il margine esterno di una metà membrana per separarlo dal supporto di plastica; evitare l&#39;interazione con le cellule raccolte sulla membrana, tra cui quella che la membrana non si piega su se stesso. </li><li> Fissare la membrana con una pinzetta prima completamente staccando dal titolare. Posizionare la membrana in etichetta provetta 2 ml contenente 500 ml di 95% (v / v) di etanolo e lasciare a fissare per un minimo di 20 minuti ad un massimo di 2 ore prima della trasformazione (paragrafo 4.2). </li><li> Immediatamente separata seconda metà di membrana (come descritto nella sezione 3.2) e prontamente procedere con la sezione 4.1. </li></ol> La colorazione 4. Campione <ol><li> Immunocytochemical colorazione <ol><li> Posizionare con cura la membrana su 35 mm fondo piatto cultura di vetro con il lato rivolto verso il basso di raccolta; garantire membrana è piatta. </li><li> Pipettare 20 ml di composizione macchia immunocitochimica assemblato nella sezione 1 a membrana. Se riccioli campione, usare puntali monouso per appiattire con attenzione la membrana spingendo i bordi verso il basso. Evitare il contatto con le zone di raccolta delle cellule. </li><li> coprire delicatamente la membrana con copertura in vetro antiscivolo. Evitare movimenti inutili di campione. Coprire con coperchio e sigillare con lab-pellicola intorno ai bordi. Non ostruire la trasparenza di piatto (in alto e in basso) e la visibilità del campione. Etichetta con pennarello laboratorio. </li><li> Immediatamente procedere con l&#39;imaging (paragrafo 5.1), poi tornare alla sezione 4.2 per continuare con colorazione citologica dell&#39;altra metà della membrana. </li></ol></li><li> citologico colorazione <ol><li> Gradualmente idratare membrana aggiungendo lentamente 500 ml di acqua distillata al tubo used per la fissazione nella sezione 3.4. Aspirare il contenuto più volte in punta della pipetta per mescolare, quindi rimuovere il contenuto. </li><li> Aggiungere 500 ml di acqua distillata. Lasciate riposare per qualche secondo, quindi rimuovere. Aggiungere 500 ml di alcian blu e lasciar riposare per 3 min. Rimuovere macchie ed eseguire 3 consecutivi acqua distillata 500 microlitri risciacqui o fino a quando il liquido risciacqua chiaro. </li><li> Aggiungere 500 ml di Hematoxylin # 1 macchia e lasciare per 3 min. Rimuovere macchie e di eseguire tre consecutivi 500 microlitri risciacqui acqua distillata o fino a quando il liquido risciacqua chiaro. Disidratare (inverso del paragrafo 4.2.1) aspirando il liquido chiaro. </li><li> Aggiungere 500 ml di Papanicolaou OG 6-macchia e lasciare per 3 min. Rimuovere macchie ed eseguire una 500 microlitri 95% (v / v) etanolo risciacquo. </li><li> Aggiungere 500 ml di Papanicolaou EA-65 macchia e lasciare per 3 min. Rimuovere macchia ed eseguire 3 consecutivi 500 microlitri 95% (v / v) di etanolo risciacqua. </li><li> Completamente disidratano utilizzando tre consecutivi 500 microlitri 100% risciacqui etanolo. utilizzando tweEzers, rimuovere il campione dalla soluzione; evitare le zone di raccolta delle cellule commoventi. </li><li> Posizionare la membrana sul vetrino; assicurare che la linea di raccolta delle cellule è parallelo o perpendicolare scorrere margine per facilitare l&#39;allineamento durante l&#39;imaging. </li><li> Applicare una goccia di 100% di etanolo e coprire con slip in vetro. Immediatamente procedere con l&#39;imaging (paragrafo 5.2). </li></ol></li></ol> Imaging 5. Campione <ol><li> Imaging fluorescenti immunocitochimiche Macchie <ol><li> Utilizzare un microscopio confocale a scansione laser (CLSM) 11 o microscopio a fluorescenza 12, dotato di una fotocamera digitale a colori, collegato a un computer che esegue un software di acquisizione delle immagini. Grazie ai campioni essendo contenuto all&#39;interno piastre di coltura, un microscopio invertito può essere necessario. </li><li> Impostare microscopio 11,12 secondo assorbimento ed emissione spettri di etidio omodimero-1 (528/617 nm maxima Ex / Em in presenza di DNA) e calceina AM (494/517 nm Ex / Em maxima). </li><li> Selezionare l&#39;ingrandimento più basso del microscopio (ad esempio, oggettiva 2.5X) e attivare il sistema di imaging digitale; osservare il campione visualizzata sul monitor del computer. </li><li> Controllare campione per le caratteristiche cellulari di interesse. </li><li> Selezionare l&#39;ingrandimento del microscopio desiderato e di acquisire le immagini utilizzando il software di imaging. </li><li> Salvare i file sia in formato di file microscopio nativo o un formato di file non compresso (ad esempio, * .raw, * .tiff). </li></ol></li><li> Imaging citologico Macchie <ol><li> Utilizzare un microscopio standard di laboratorio in campo chiaro senza filtri, dotato di una fotocamera digitale a colori, collegato a un computer che esegue un software di acquisizione delle immagini. </li><li> Luogo e vetrino di blocco (dal punto 4.2.8) sul palco microscopio. Reidratare campione con 100% di etanolo come necessario, durante l&#39;intera procedura di imaging. </li><li> Selezionare l&#39;ingrandimento più basso del microscopio (ad esempio, oggettiva 2.5X) e attivare °sistema di imaging digitale e; osservare il campione visualizzata sul monitor del computer. </li><li> raccolta delle cellule Garantire è allineato con entrambi gli assi X o Y del controllo palco microscopio. Regolare, se necessario, rimuovendo il vetrino e ruotando il campione con un puntale o di una pinzetta. </li><li> Utilizzando il software di imaging, cattura di immagini (s) dell&#39;intero campione con il più basso di ingrandimento del microscopio. </li><li> Passare a un ingrandimento al microscopio moderata (ad esempio, 10 - 20X obiettivo) e regolare fonte di luce microscopio e parametri di imaging software (esposizione, contrasto, bilanciamento del bianco, ecc), e disabilitare il controllo del software automatizzato. Salvare queste impostazioni per un utilizzo futuro. </li><li> Determinare inizio e fine i punti di scansione (ad esempio, in alto a sinistra e angoli basso a destra della raccolta delle cellule). </li><li> Utilizzando il software di imaging, cattura di immagini (s) di tutta l&#39;area di raccolta delle cellule all&#39;interno dei punti iniziale e finale. Garantire un 20% side-to-side e top-to-bottom sovrapposizione between tutte le immagini adiacenti. </li><li> Salvare i file utilizzando il formato di file nativo del microscopio o un formato di file non compresso (ad esempio, * .raw, * .tiff). </li><li> Genera immagine panoramica finale utilizzando software automatici di cucitura di scelta (ad esempio, Adobe Photoshop Elements). Utilizzare immagini di riferimento prese a passo 5.2.5) per confermare l&#39;accuratezza delle cuciture automatizzata nell&#39;immagine finale. </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.

Citologia ad impressione viene in genere eseguita sulla congiuntiva bulbare, utilizzando cellulosa misto membrane estere. I campioni sono tagliati a misura da fogli di materiale alla rinfusa, sterilizzati, applicati e rimossi dalla superficie congiuntivale con pinze. Utilizzando lo stesso materiale della membrana, un dispositivo IC pistone controllato è stato recentemente progettato per abbinare la geometria della superficie della congiuntiva bulbare, e mantenere la pressione costante tra le applicazioni 13....

La palpebra superiore umano esegue circa 10.000 lampeggia ogni giorno 1, con appena un 1 - regione congiuntivale due millimetri stretta opposti del globo oculare. Grazie al suo moto di pulitura durante il lampeggio, questa porzione del margine coperchio è stata definita la regione &quot;coperchio tergicristallo&quot; 2. Si presume che l&#39;attrito aumenta in questa regione durante lampeggiamento abituale, grazie al coperchio pulire il mondo. Questo può svolgere un ruolo significativo in termini di comfort oculare. Di lenti a contatto, in particolare, l&#39;esperienza disagio oculare e questo è uno dei motivi principali per cessare l&#39;obiettivo di usura 3. Quando una lente a contatto è posto sugli occhi, il coefficiente di attrito tra il materiale della lente e la superficie oculare ha dimostrato di cambiare 4. Ci sono prove che suggeriscono che i sintomi di secchezza potrebbero essere correlati a questo attrito alterato 2,5,6. Questa associazione può anche tradursi in fenomeni osservabili clinicamente, in particolare coperchio wiper epiteliopatia (LWE), chiamato anche palpebra superiore margine di colorazione (ULMs) 6. LWE è un segno precoce di irritazione oculare e un potenziale fattore predittivo per la malattia dell&#39;occhio secco. Si è osservato e misurato dalla colorazione vitale delle regioni margine coperchio superiore e inferiore. Anche se questo sistema di classificazione 2 e la sua validità clinica (ad esempio, la correlazione con i sintomi soggettivi) sono ancora in discussione, fastidio oculare rimane un enigma per i medici e gli scienziati allo stesso modo e, soprattutto, un inconveniente per i pazienti. Ad oggi, poco si sa circa le variazioni clinicamente rilevanti nel (sotto) l&#39;anatomia e la fisiologia cellulare del coperchio zona tergicristallo 7 e un solo rapporto fa uso di metodi citologici 8. Citologia ad impressione (IC) è stato impiegato per più di 40 anni per raccogliere le cellule dalla superficie epiteliale del bulbare o congiuntiva tarsale mediante l&#39;applicazione di una membrana 9,10. Dopo la rimozione, le cellule aderenti subiscono ccolorazione ytological e di imaging microscopico. A causa delle differenze anatomiche e cellulari della regione tergicristallo coperchio, questa tecnica richiede l&#39;ottimizzazione.

<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.

La membrana coltura cellulare attraversato tutto il supporto di plastica è un comodo strumento per la raccolta portatile delle cellule epiteliali della congiuntiva coperchio tergicristallo. Questo metodo elimina la necessità di strumenti sterilizzati supplementari tipicamente usati per preparare e gestire membrane IC. Figura 1 illustra la IC dell&#39;area tergicristallo coperchio usando un inserto di coltura cellulare. Abbiamo ottimizzato due diversi protocolli di colo...

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.

Citologia ad impressione del coperchio Area Wiper

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 ...

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10.3K Views.  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. 

Video: 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.

Rilevamento e Genogrouping di Norovirus dalle feci dei bambini 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 ...

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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.

Colorazione immunoistochimica di B7-H1 (PD-L1) in paraffina incorporate diapositive di tessuto pancreatico Adenocarcinoma

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 Imaging di fluorescenza biventricolare isolato lavoro Hearts Coniglio

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 identificazione dei singoli HERV Loci Expression: Domanda di Biomarker Discovery in Cancro alla prostata

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.

Raccolta ed estrazione di Saliva DNA per il sequenziamento di nuova generazione

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.

A &quot;paziente-Like&quot; ortotopico Singenico modello murino della carcinoma epatocellulare Metastasi

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.

Induzione di ictus ischemico e di ischemia-riperfusione nei topi mediante la tecnica di occlusione dell&#39;arteria Medio e visualizzazione di Infarct Area

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.

Sintesi radiochimica automatizzata di [<sup&gt; 18</sup&gt; F] 3F4AP: un nuovo tracciante PET per le malattie Demyelinating di imaging

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 Cytologia del Tubo Fallopiano come strumento promettente per il rilevamento precoce del cancro ovarico

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.

Accesso alla citotossicità e alla risposta cellulare ai biomateriali

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

Citologia ad impressione del coperchio Area Wiper

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