Name: four-color fluorescence immunohistochemistry of t-cell subpopulations in archival formalin-fixed, paraffin-embedded human oropharyngeal squamous cell carcinoma samples
Uploaded: 2017-07-29T14:00:00
Description: Watch this Scientific Journal Video about Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinom

Simone Punt blev støttet af grant UL2010-4801 fra det hollandske Cancer Society. Vi vil gerne takke alle medforfatterne af det originale papir, at denne JoVE-protokol er baseret på: Emilie A. Dronkers, Marij JP Welters, Renske Goedemans, Senada Koljenović, Elisabeth Bloemena, Peter JF Snijders, Arko Gorter og Sjoerd van Der Burg.

Patientprøver blev behandlet i henhold til de medicinske etiske retningslinjer, der er beskrevet i adfærdskodeksen for korrekt sekundær anvendelse af humant væv fra den nederlandske sammenslutning af biomedicinske videnskabelige samfund (www.federa.org). 1. Klargør dias <ol><li> Hent resekteret vævsmateriale (enhver form for væv) fra en patologi afdeling efter at have fået medicinsk etisk udvalg tilladelse til at anvende resekteret materiale. BEMÆRK: For det nuværende eksp...

<ol>
	<li>Westra, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+changing+face+of+head+and+neck+cancer+in+the+21st+century:+the+impact+of+HPV+on+the+epidemiology+and+pathology+of+oral+cancer.">The changing face of head and neck cancer in the 21st century: the impact of HPV on the epidemiology and pathology of oral cancer.</a> Head Neck Pathol. 3 (1), 78-81 (2009).</li><li>Rietbergen, M. M., 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=Human+papillomavirus+detection+and+comorbidity:+critical+issues+in+selection+of+patients+with+oropharyngeal+cancer+for+treatment+De-escalation+trials.">Human papillomavirus detection and comorbidity: critical issues in selection of patients with oropharyngeal cancer for treatment De-escalation trials.</a> Ann Oncol. 24 (11), 2740-2745 (2013).</li><li>Wallis, S. P., Stafford, N. D., Greenman, 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+relevance+of+immune+parameters+in+the+tumor+microenvironment+of+head+and+neck+cancers.">Clinical relevance of immune parameters in the tumor microenvironment of head and neck cancers.</a> Head Neck. 37 (3), 449-459 (2015).</li><li>Ye, J., Livergood, R. S., Peng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+and+regulation+of+human+Th17+cells+in+tumor+immunity.">The role and regulation of human Th17 cells in tumor immunity.</a> Am J Pathol. 182 (1), 10-20 (2013).</li><li>Amedei, A., 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=Ex+vivo+analysis+of+pancreatic+cancer-infiltrating+T+lymphocytes+reveals+that+ENO-specific+Tregs+accumulate+in+tumor+tissue+and+inhibit+Th1/Th17+effector+cell+functions.">Ex vivo analysis of pancreatic cancer-infiltrating T lymphocytes reveals that ENO-specific Tregs accumulate in tumor tissue and inhibit Th1/Th17 effector cell functions.</a> Cancer Immunol Immunother. 62 (7), 1249-1260 (2013).</li><li>Whiteside, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+are+regulatory+T+cells+(Treg)+regulating+in+cancer+and+why?.">What are regulatory T cells (Treg) regulating in cancer and why?.</a> Semin Cancer Biol. 22 (4), 327-334 (2012).</li><li>Punt, S., 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=Angels+and+demons:+Th17+cells+represent+a+beneficial+response,+while+neutrophil+IL-17+is+associated+with+poor+prognosis+in+squamous+cervical+cancer.">Angels and demons: Th17 cells represent a beneficial response, while neutrophil IL-17 is associated with poor prognosis in squamous cervical cancer.</a> Oncoimmunology. 4 (1), e984539 (2015).</li><li>Bamias, A., 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=Significant+differences+of+lymphocytes+isolated+from+ascites+of+patients+with+ovarian+cancer+compared+to+blood+and+tumor+lymphocytes.+Association+of+CD3+CD56++cells+with+platinum+resistance.">Significant differences of lymphocytes isolated from ascites of patients with ovarian cancer compared to blood and tumor lymphocytes. Association of CD3+CD56+ cells with platinum resistance.</a> Gynecol Oncol. 106 (1), 75-81 (2007).</li><li>Gasparoto, T. H., 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=Patients+with+oral+squamous+cell+carcinoma+are+characterized+by+increased+frequency+of+suppressive+regulatory+T+cells+in+the+blood+and+tumor+microenvironment.">Patients with oral squamous cell carcinoma are characterized by increased frequency of suppressive regulatory T cells in the blood and tumor microenvironment.</a> Cancer Immunol Immunother. 59 (6), 819-828 (2010).</li><li>Jordanova, E. S., 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=Human+leukocyte+antigen+class+I,+MHC+class+I+chain-related+molecule+A,+and+CD8+/regulatory+T-cell+ratio:+which+variable+determines+survival+of+cervical+cancer+patients?.">Human leukocyte antigen class I, MHC class I chain-related molecule A, and CD8+/regulatory T-cell ratio: which variable determines survival of cervical cancer patients?.</a> Clin Cancer Res. 14 (7), 2028-2035 (2008).</li><li>van Esch, E. M., 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=Alterations+in+classical+and+nonclassical+HLA+expression+in+recurrent+and+progressive+HPV-induced+usual+vulvar+intraepithelial+neoplasia+and+implications+for+immunotherapy.">Alterations in classical and nonclassical HLA expression in recurrent and progressive HPV-induced usual vulvar intraepithelial neoplasia and implications for immunotherapy.</a> Int J Cancer. 135 (4), 830-842 (2014).</li><li>Heeren, A. M., 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=Prognostic+effect+of+different+PD-L1+expression+patterns+in+squamous+cell+carcinoma+and+adenocarcinoma+of+the+cervix.">Prognostic effect of different PD-L1 expression patterns in squamous cell carcinoma and adenocarcinoma of the cervix.</a> Mod Pathol. 29 (7), 753-763 (2016).</li><li>Punt, S., 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=A+beneficial+tumor+microenvironment+in+oropharyngeal+squamous+cell+carcinoma+is+characterized+by+a+high+T+cell+and+low+IL-17(+)+cell+frequency.">A beneficial tumor microenvironment in oropharyngeal squamous cell carcinoma is characterized by a high T cell and low IL-17(+) cell frequency.</a> Cancer Immunol Immunother. 65 (4), 393-403 (2016).</li><li>Kirkwood, B. R., Sterne, J. A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Essential Medical Statistics. , (2003).</li><li>Kleinbaum, D. G., Klein, 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> Survival Analysis: A Self-Learning Text. , (2005).</li><li>Fabre, J., 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=Targeting+the+Tumor+Microenvironment:+The+Protumor+Effects+of+IL-17+Related+to+Cancer+Type.">Targeting the Tumor Microenvironment: The Protumor Effects of IL-17 Related to Cancer Type.</a> Int J Mol Sci. 17 (9), (2016).</li><li>Feng, Z., 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=Multispectral+imaging+of+formalin-fixed+tissue+predicts+ability+to+generate+tumor-infiltrating+lymphocytes+from+melanoma.">Multispectral imaging of formalin-fixed tissue predicts ability to generate tumor-infiltrating lymphocytes from melanoma.</a> J Immunother Cancer. 3 (47), (2015).</li></ol>

For den beskrevne protokol er et af de mest kritiske trin at bestemme den korrekte fortynding af de anvendte primære antistoffer. Fortyndingen af ​​de mærkede sekundære antistoffer var 1: 200, som anbefalet af producenten af ​​disse specifikke Alexa-mærkede antistoffer. Fortyndingen af ​​de primære antistoffer bør derefter bestemmes ved en seriel fortynding, fortrinsvis under anvendelse af to forskellige prøver af den tilsigtede vævstype (i dette tilfælde OPSCC). Den optimale arbejdsfortynding er fo...

Oropharyngeal squamous cell carcinomas (OPSCCs) er en heterogen gruppe af pladecellercancer med oprindelse i oropharynx. Risikofaktorer for OPSCC omfatter humant papillomavirus (HPV) infektion og alkohol og tobaksbrug 1 , 2 . Immunresponsens rolle og hvordan man bruger det i en klinisk indstilling, er lige begyndt at blive udforsket. Tumormiljømiljøet infiltreres af immunceller, der tiltrækkes til kræftstedet. Skønt en høj CD8 + cytotoksisk T-cellefrekvens er korreleret med forbedret overlevelse hos OPSCC-patienter 3 , er rollen af ​​andre T-celle-subsæt, herunder Tregs og Th17-celler, stadig uklar 4 . Mens Th1- og Th17-celler skal hjælpe med immunresponsmålrettede tumorceller, er Tregs velkendte for deres evner til at undertrykke aktiviteten af ​​andre T-celler 5 . Tilstedeværelsen af ​​TRegs har vist sig at korrelere med både gunstige og ugunstige responser i forskellige tumortyper 6 . Da ikke alle immunceller, der er til stede i blodet, infiltrerer tumoren i samme grad, giver undersøgelsen af ​​det lokale tumormikromiljø det mest pålidelige mål for immunrespons rettet mod tumoren. Formålet med denne undersøgelse er at bestemme sammenhængen mellem antal og typer af immunceller og det kliniske resultat. Vi anvendte firefarvet fluorescens IHC-billeddannelse til at analysere antallet og lokaliseringen af ​​forskellige T-celle subpopulationer i human OPSCC. Vi fokuserede på samlede T-lymfocytter (CD3 + ), Th17-celler og immunosuppressive FoxP3 + Tregs, hvis differentieringsveje er nært beslægtet med Th17-celler. Th17-celler er kendetegnet ved kombinationen af ​​CD3 og IL-17. Cytokinet IL-17 kan også fremstilles af ikke-T-celler 7 . Vi fastslog fordelingen af ​​intraepitHelial- og strom-T-celler, Tregs, Th17 og IL-17 + ikke-T-celler i en stor serie af OPSCC-tilfælde og analyserede korrelationerne med patientoverlevelse. Multicolor fluorescens IHC blev brugt til at identificere ekspressionen af ​​CD3, Foxp3 og IL-17 i kombination med en DAPI-modstregning. Dette assay tillod for nem og klar identifikation af begge tumorceller (ved anvendelse af DAPI-nuklearfarvning) og de infiltrerende T-cellepopulationer (ved anvendelse af en kombination af forskellige markører). Efter prøvepræparation og farvning blev et fluorescerende mikroskop og billeddannelsessoftware anvendt til at adskille de forskellige fluorescerende farver, der blev brugt, og til at bestemme antallet og typen af ​​celler, der er til stede i både tumorepitelet og den tumorassocierede stroma. Et alternativt assay til at kvantificere og fænotype immuncellepopulationer er flowcytometrianalyse eller cytometri ved flygtid (CyTOF) analyse af tumor eller perifere prøver ( dvs. blod eller ascites). Brug detteTeknik, tabes al information om lokalisering og relativ fordeling af de forskellige celletyper. Anvendelsen og analysen af ​​perifere prøver giver heller ikke oplysninger om hvilke celler der er i stand til at infiltrere tumormikromiljøet. Blod- og asciteimmuncelleanalyser har vist sig ikke at reflektere fænotypen og hyppigheden af ​​immuncelleinfiltrering af tumorvævet 8 , 9 . Et andet alternativ er brugen af ​​lysfeltmikroskopi. En fordel ved denne teknik over fluorescensbilleddannelse er fraværet af vævsautofluorescens. Selvom nogle prøver indeholder mere autofluorescens-især erythrocytter, men også andre celletyper, herunder neutrofile granulocytter, kan disse områder let fjernes i analysen af ​​næsten alle prøver. Immunofluorescens giver fordelen ved at analysere flere markører i en prøve ved anvendelse af et panel med målrettet fluoresceNt bølgelængder. Dette er i øjeblikket umuligt i samme omfang for lysfeltmikroskopi på grund af manglen på et tilstrækkeligt antal mærker og kommercielt tilgængelige antistofisotyper for et bestemt antigen. Den multicolor fluorescens IHC-teknik, der er beskrevet her, er blevet anvendt i mange forskellige kræfttyper og antistofkombinationer til at studere forskellige immuncellepopulationer såvel som tumorcelle-udtrykte molekyler, såsom humane leukocytantigener (HLA) og PD-L1 10 , 11 , 12 . Protokollen er blevet etableret og valideret ved hjælp af mange forskellige typer af prøver og antistoffer.

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			<td height="21" style="height:21px;width:375px;">Pathos Delta Ultra Rapid Tissue Processor</td>
			<td style="width:167px;">Milestone and Histostar</td>
			<td style="width:143px;"></td>
			<td style="width:696px;">Automated tissue processor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Histostar</td>
			<td style="width:167px;">Thermo Scientific&#160;</td>
			<td style="width:143px;"></td>
			<td style="width:696px;">Tissue block embedding machine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Formaldehyde</td>
			<td style="width:167px;">Baker</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Xylol</td>
			<td style="width:167px;">Merck</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Ethanol</td>
			<td style="width:167px;">Merck</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">milliQ water</td>
			<td style="width:167px;">Elaga Purelab Chorus</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Paraffin wax/Paraclean</td>
			<td style="width:167px;">Klinipath</td>
			<td style="width:143px;">5079A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Microtome tissue holder&#160;</td>
			<td style="width:167px;">Leica</td>
			<td style="width:143px;">RM225</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flex IHC side</td>
			<td style="width:167px;">Dako</td>
			<td style="width:143px;"></td>
			<td>Tissue slide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Tris</td>
			<td style="width:167px;">Merk-Milipore</td>
			<td style="width:143px;">1,083,821,000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">EDTA</td>
			<td style="width:167px;">Baker</td>
			<td style="width:143px;">1073</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">PBS</td>
			<td style="width:167px;">Bio-Rad</td>
			<td>BUF036A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">BSA</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:143px;">A9647</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Rabbit anti-CD3</td>
			<td style="width:167px;">Abcam</td>
			<td style="width:143px;">ab828</td>
			<td>Titrate required antibody dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Mouse IgG1 anti-FoxP3</td>
			<td style="width:167px;">Abcam</td>
			<td style="width:143px;">ab20034</td>
			<td>Titrate required antibody dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Goat IgG anti-IL-17</td>
			<td style="width:167px;">R&#38;D Systems</td>
			<td style="width:143px;">AF-317-NA</td>
			<td>Titrate required antibody dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Rabbit Ig isotype control antibody</td>
			<td style="width:167px;">Abcam</td>
			<td>ab27472</td>
			<td>Use at same final concentration as anti-CD3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Mouse IgG1 isotype control antibody</td>
			<td style="width:167px;">Abcam</td>
			<td>ab91353</td>
			<td>Use at same final concentration as anti-FoxP3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Goat IgG isotype control antibody</td>
			<td style="width:167px;">ThermoFisher Scientific</td>
			<td style="width:143px;">02-6202</td>
			<td>Use at same final concentration as anti-IL-17</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Donkey anti-rabbit IgG A546</td>
			<td style="width:167px;">ThermoFisher Scientific</td>
			<td style="width:143px;">A10040</td>
			<td>Dilute 1:200 in 1% BSA/PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Donkey anti-mouse-A647</td>
			<td style="width:167px;">ThermoFisher Scientific</td>
			<td style="width:143px;">A31571</td>
			<td>Dilute 1:200 in 1% BSA/PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Donkey anti-goat IgG A488</td>
			<td style="width:167px;">ThermoFisher Scientific</td>
			<td style="width:143px;">A11055</td>
			<td>Dilute 1:200 in 1% BSA/PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">VectaShield containing DAPI</td>
			<td style="width:167px;">Vector Laboratories</td>
			<td style="width:143px;">H-1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">LSM700 confocal laser scanning microscope&#160;</td>
			<td style="width:167px;">Zeiss</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">LCI Plan-Neofluar 25x/0.8 Imm Korr DIC M27 objective</td>
			<td style="width:167px;">Zeiss</td>
			<td>420852-9972-720</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">LSM Zen Software</td>
			<td style="width:167px;">Zeiss</td>
			<td>version 2009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">LSM Image Browser</td>
			<td style="width:167px;">Zeiss</td>
			<td style="width:143px;">version 4.2.0.121</td>
			<td>Available to download at www.zeiss.com/microscopy/int/website/downloads/lsm-image-browser.html.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">SPSS</td>
			<td style="width:167px;">IBM Corp.</td>
			<td style="width:143px;">version 20.0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">ImageJ</td>
			<td style="width:167px;"></td>
			<td style="width:143px;">version 1.50i</td>
			<td>Available to download at http://rsb.info.nih.gov/ij.</td>
		</tr>
	</tbody>
</table>

four-color fluorescence immunohistochemistry of t-cell subpopulations in archival formalin-fixed, paraffin-embedded human oropharyngeal squamous cell carcinoma samples

Den firefarvede fluorescens immunhistokemi (IHC) teknik er en metode til at kvantificere cellepopulationer af interesse under hensyntagen til deres relative fordeling og deres lokalisering i vævet. Denne teknik er blevet anvendt i stor udstrækning for at studere immuninfiltreret i forskellige tumortyper. Tumormiljømiljøet infiltreres af immunceller, som tiltrækkes af tumorstedet. Forskellige immuncellepopulationer har vist sig at spille forskellige roller i tumormikromiljøet og at have en anden indflydelse på sygdommens udfald. Dette manuskript beskriver brugen af ​​multiparameter fluorescens IHC på oropharyngeal squamous cell carcinoma (OPSCC) som et eksempel. Denne teknik kan udvides til andre vævsprøver og celletyper af interesse. I den præsenterede undersøgelse analyserede vi det intraepiteliale og stromale rum i en stor OPSCC kohorte (n = 162). Vi fokuserede på samlede T-lymfocytter (CD3 + ), immunosuppressive regulatOry-T-celler (Tregs, dvs. FoxP3 + ) og T-hjælper 17 (Th17) celler ( dvs. IL-17 + CD3 + ) ved anvendelse af et nukleært modstykke for at skelne tumorepitel fra stroma. Et stort antal T-celler viste sig at være korreleret med forbedret sygdomsfri overlevelse hos patienter med et lavt antal intratumorale IL-17 + ikke-T-celler. Dette antyder, at IL-17 + ikke-T-celler kan korreleres med en dårlig immunrespons i OPSCC, hvilket er i overensstemmelse med korrelationen beskrevet mellem IL-17 og dårlig overlevelse hos cancerpatienter. I øjeblikket udvikles nye multifarameterfluorescens IHC-teknikker ved anvendelse af op til 7 forskellige fluorokromer og vil muliggøre mere præcis karakterisering og lokalisering af immunceller i tumormikromiljøet.

En række FFPE-forbehandlings-tumorprøver opnået fra primære oropharyngeale tumorer, der blev diagnosticeret i Leiden University Medical Center, Leiden, Holland mellem 1970 og 2011, valgt som beskrevet tidligere (n = 162) blev farvet ved anvendelse af den beskrevne protokol 13 . En til fire tilfældige billeder af hvert dias blev analyseret ( figur 1 ). Nogle autofluorescerende celler er angivet, hvilket klart kan skelnes mellem de...

Watch this Scientific Journal Video about Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinom

Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinoma Samples

Multiparameterfluorescensimmunohistokemi kan anvendes til at vurdere antallet, den relative fordeling og lokalisering af immuncellepopulationer i tumormikromiljøet. Dette manuskript beskriver brugen af ​​denne teknik til analyse af T-celle subpopulationer i orofaryngealkræft.

Four-Color Fluorescence Immunohistochemistry af T-Cell Subpopulations i Archival Formalin-Fixed, Paraffin-Embedded Human Oropharyngeal Squamous Cell Carcinoma Samples

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their ...

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Cancer Research

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