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.

Ethics statement: Prior to collecting cells from human subjects, informed consent and ethics approval must be obtained.
1. Prepare Stain
NOTE: Prepare stain on day of experiment.
<ol>
	<li>Add 5 &#181;l Ethidium (or 4 &#181;M) and 5 &#181;l Calcein AM (or 4 &#181;M) to 2.5 ml phosphate buffered saline (PBS) in 15 ml centrifuge tube; mix. Wrap in aluminum foil to shield from light and store at RT until use.</li>
</ol>
2. Collect Cells
<ol>
	<li>Remove cell culture inserts from their package and label for each eyelid to be sampled. Mark orientation of cell collection on the side of the membrane plastic holder using permanent marker. Conduct slit lamp inspection at moderate magnification (approximately 20X) to confirm appropriate health of the region to undergo IC.</li>
	<li>Dispense one drop of topical anesthetic (0.5% proparacaine hydrochloride) in the lower conjunctival sac of each eye and instruct participant to keep eyes closed for one minute.</li>
	<li>Evert upper eye lid and hold in place by the lashes; avoid any contact with the lid wiper area. 
	Optional: the aid of a secondary investigator may be required to perform this step.</li>
	<li>Apply membrane perpendicularly onto the central part of the lid wiper region with minimal pressure.</li>
	<li>Hold membrane in place for 3 - 5 sec. Observe the membrane become translucent in the contact area. At this point, gently remove the membrane and permit the participant&#39;s lid to &#34;flip&#34; back into place.</li>
	<li>Promptly apply one drop of PBS to the sample, to prevent it from drying out. Membrane will turn translucent and should not turn opaque at any time, as this indicates drying. Have the secondary investigator proceed immediately with processing of samples (section 3).</li>
	<li>Conduct final eye check to ensure integrity of the conjunctiva. Dispense ocular lubricants and instruct participant to avoid rubbing their eyes until anesthetic effect wears off (approx. 15 min).</li>
</ol>
3. Sample Processing
<ol>
	<li>Using micro-scissors, cut the membrane in half (one half for each of the downstream analyses), perpendicular through the middle of the cell collection. Cut along the outer margin of one membrane half to separate it from the plastic holder; avoid interaction with the collected cells on the membrane, including ensuring that the membrane does not fold onto itself.</li>
	<li>Secure the membrane using tweezers before completely detaching from holder. Place membrane into labelled 2 ml centrifuge tube containing 500 &#181;l of 95% (v/v) ethanol and leave to fix for a minimum of 20 min to a maximum of 2 hr before processing (section 4.2).</li>
	<li>Immediately separate second half of membrane (as described in section 3.2) and promptly proceed with section 4.1.</li>
</ol>
4. Sample Staining
<ol>
	<li>Immunocytochemical Staining
	<ol>
		<li>Carefully place membrane in 35 mm glass bottom culture dish with collection side facing down; ensure membrane is flat.</li>
		<li>Pipette 20 &#181;l of immunocytochemical stain composition assembled in section 1 on to the membrane. If sample curls up, use disposable pipette tips to carefully flatten the membrane by pushing its edges down. Avoid contact with cell collection areas.</li>
		<li>Gently cover the membrane with glass cover slip. Avoid unnecessary movement of sample. Cover dish with lid and seal with lab-film around the edges. Do not obstruct transparency of dish (top and bottom) and visibility of sample. Label with lab marker.</li>
		<li>Immediately proceed with imaging (section 5.1), then return to section 4.2 to continue with cytological staining of the other half of the membrane.</li>
	</ol>
	</li>
	<li>Cytological Staining
	<ol>
		<li>Gradually hydrate membrane by slowly adding 500 &#181;l of distilled water to the tube used for fixation in section 3.4. Aspirate contents several times into pipette tip to mix, then remove contents.</li>
		<li>Add 500 &#181;l of distilled water. Let stand for a few seconds, then remove. Add 500 &#181;l of Alcian Blue stain and leave for 3 min. Remove stain and perform 3 consecutive 500 &#181;l distilled water rinses or until liquid rinses clear.</li>
		<li>Add 500 &#181;l of Hematoxylin # 1 stain and leave for 3 min. Remove stain and perform three consecutive 500 &#181;l distilled water rinses or until liquid rinses clear. Dehydrate (reverse of section 4.2.1) by aspirating the clear liquid.</li>
		<li>Add 500 &#181;l of Papanicolaou OG-6 stain and leave for 3 min. Remove stain and perform one 500 &#181;l 95% (v/v) ethanol rinse.</li>
		<li>Add 500 &#181;l of Papanicolaou EA-65 stain and leave for 3 min. Remove stain and perform 3 consecutive 500 &#181;l 95% (v/v) ethanol rinses.</li>
		<li>Fully dehydrate using three consecutive 500 &#181;l 100% ethanol rinses. Using tweezers, remove sample from solution; avoid touching cell collection areas.</li>
		<li>Place membrane on glass slide; ensure that cell collection line is either parallel or perpendicular to slide margin for easier alignment during imaging.</li>
		<li>Apply a drop of 100% ethanol and cover with glass slip. Immediately proceed with imaging (section 5.2).</li>
	</ol>
	</li>
</ol>
5. Sample Imaging
<ol>
	<li>Imaging Fluorescent Immunocytochemical Stains
	<ol>
		<li>Use a confocal laser scanning microscope (CLSM)11 or fluorescence microscope12, equipped with a digital color camera, connected to a computer running an image acquisition software. Due to the samples being contained within culture dishes, an inverted microscope may be necessary.</li>
		<li>Set up microscope11,12 according to absorption and emission spectra of ethidium homodimer-1 (528/617 nm Ex/Em maxima in the presence of DNA) and calcein AM (494/517 nm Ex/Em maxima).</li>
		<li>Select the microscope&#39;s lowest magnification (e.g., 2.5X objective) and activate the digital imaging system; observe the sample displayed on the computer monitor.</li>
		<li>Inspect sample for cellular features of interest.</li>
		<li>Select desired microscope magnification and acquire images using imaging software.</li>
		<li>Save files in either native microscope file format or an uncompressed file format (e.g., *.raw, *.tiff).</li>
	</ol>
	</li>
	<li>Imaging Cytological Stains
	<ol>
		<li>Use a standard laboratory bright-field microscope with no filters, equipped with a digital color camera, connected to a computer running an image acquisition software.</li>
		<li>Place and lock glass slide (from step 4.2.8) on microscope stage. Rehydrate sample with 100% ethanol as needed, throughout the entire imaging procedure.</li>
		<li>Select the microscope&#39;s lowest magnification (e.g., 2.5X objective) and activate the digital imaging system; observe the sample displayed on the computer monitor.</li>
		<li>Ensure cell collection is aligned with either X or Y axis of microscope stage control. Adjust if needed, by removing the cover slip and rotating the sample using a pipette tip or tweezers.</li>
		<li>Using the imaging software, capture image(s) of the entire sample with the lowest microscope magnification.</li>
		<li>Switch to moderate microscope magnification (e.g., 10 - 20X objective) and adjust microscope light source and software imaging parameters (exposure, contrast, white balance, etc.), and disable their automated software metering. Save these settings for future use.</li>
		<li>Determine beginning and end points of scan (e.g., top left and bottom right corners of cell collection).</li>
		<li>Using the imaging software, capture image(s) of the entire cell collection area within the starting and end points. Ensure a 20% side-to-side and top-to-bottom overlap between all adjacent images.</li>
		<li>Save files using the native microscope file format or an uncompressed file format (e.g., *.raw, *.tiff).</li>
		<li>Generate final panoramic image using automated stitching software of choice (e.g., Adobe Photoshop Elements). Use reference images taken at step 5.2.5) to confirm the accuracy of the automated stitching in the final image.</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.

Impression cytology is typically performed on the bulbar conjunctiva, using mixed cellulose ester membranes. Samples are cut to size from bulk material sheets, sterilized, applied and removed from the conjunctival surface using forceps. Using the same membrane material, a piston-controlled IC device was recently designed to match the surface geometry of the bulbar conjunctiva, and maintain consistent pressure between applications13. These approaches are inefficient for the narrow, prominently curved lid wiper ...

The human upper eyelid executes around 10,000 blinks every day1, with just a 1 - 2 mm narrow conjunctival region opposing the ocular globe. Due to its wiping motion during the blink, this portion of the lid margin has been termed the &#34;lid wiper&#34; region2. It is assumed that friction is increased in this region during habitual blinking, due to the lid wiping over the globe. This may play a significant role in ocular comfort. Contact lens wearers in particular experience ocular discomfort and this is one of the primary reasons for ceasing lens wear3. When a contact lens is placed on the eye, the friction coefficient between the lens material and the ocular surface has been shown to change4. There is evidence to suggest that dryness symptoms could be related to this altered friction2,5,6.
This association may also be reflected in clinically observable phenomena, notably lid wiper epitheliopathy (LWE), also called upper lid margin staining (ULMS)6. LWE is an early sign of ocular irritation and a potential predictor for dry eye disease. It is observed and measured by the vital staining of the upper and lower lid margin regions. While this grading system2 and its clinical validity (i.e., correlation with subjective symptoms) are still under debate, ocular discomfort remains a conundrum for clinicians and scientists alike and, most importantly, an inconvenience for patients.
To-date, little is known about clinically-relevant variations in the (sub-)cellular anatomy and physiology of the lid wiper area7 and only one report makes use of cytological methods8. Impression cytology (IC) has been employed for over 40 years to collect cells from the epithelial surface of the bulbar or tarsal conjunctiva by application of a membrane9,10. Upon removal, the adherent cells undergo cytological staining and microscopic imaging. Due to the anatomic and cellular differences in the lid wiper region, this technique requires optimization.

<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.

The cell culture membrane spanned over the plastic holder is a convenient tool for handheld collection of epithelial cells from the lid wiper conjunctiva. This method eliminates the need for additional sterilized instruments typically used to prepare and handle IC membranes. Figure 1 depicts the IC of the lid wiper area using a cell culture insert. We optimized two different staining protocols that complement each other to characterize epithelial cells from the lid wiper ...

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.

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

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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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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