Simone Punt was supported by grant UL2010-4801 from the Dutch Cancer Society. We would like to thank all the co-authors of the original paper that this JoVE protocol is based on: Emilie A. Dronkers, Marij J. P. Welters, Renske Goedemans, Senada Koljenovi&#263;, Elisabeth Bloemena, Peter J. F. Snijders, Arko Gorter, and Sjoerd van der Burg.

Patient samples were handled according to the medical ethical guidelines described in the Code of Conduct for Proper Secondary Use of Human Tissue of the Dutch Federation of Biomedical Scientific Societies (www.federa.org).
1. Prepare Slides
<ol>
	<li>Obtain resected tissue material (any kind of tissue) from a pathology department after obtaining medical ethical committee permission to use resected material. 
	NOTE: For the current experiment, pretreatment tumor samples were obtained fr...

<ol>
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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>

The authors declare no commercial or financial conflict of interest.

For the protocol described, one of the most critical steps is to determine the correct dilution of the primary antibodies used. The dilution of the labeled secondary antibodies was 1:200, as recommended by the manufacturer of these specific Alexa-labeled antibodies. The dilution of the primary antibodies should then be determined by a serial dilution, preferably using two different samples of the intended tissue type (in this case, OPSCC). The optimal working dilution is the dilution at which a clear signal is obtained, ...

Oropharyngeal squamous cell carcinomas (OPSCCs) are a heterogeneous group of squamous cell cancers originating in the oropharynx. Risk factors for OPSCC include human papillomavirus (HPV) infection and alcohol and tobacco use1,2. The role of the immune response and how to use this in a clinical setting is just starting to be explored. The tumor microenvironment is infiltrated by immune cells that are attracted to the cancer site. Although a high CD8+ cytotoxic T-cell frequency has been correlated with improved survival in OPSCC patients3, the role of other T-cell subsets, including Tregs and Th17 cells, is still unclear4. Whereas Th1 and Th17 cells are supposed to aid in the immune response targeting tumor cells, Tregs are well known for their abilities to suppress the activity of other T cells5. However, the presence of Tregs has been found to correlate with both favorable and unfavorable responses in different tumor types6. Since not all immune cells present in the blood infiltrate the tumor to the same extent, studying the local tumor microenvironment provides the most reliable measure of the immune response directed against the tumor. The aim of this study is to determine the correlation between the numbers and types of immune cells and the clinical outcome. We used four-color fluorescence IHC imaging to analyze the number and localization of various T-cell subpopulations in human OPSCC.
We focused on total T lymphocytes (CD3+), Th17 cells, and immunosuppressive FoxP3+ Tregs, whose differentiation pathway is closely related to Th17 cells. Th17 cells are characterized by the combination of CD3 and IL-17. The cytokine IL-17 can also be produced by non-T cells7. We determined the distribution of intra-epithelial and stromal T cells, Tregs, Th17, and IL-17+ non-T cells in a large series of OPSCC cases and analyzed the correlations with patient survival. Multicolor fluorescence IHC was used to identify the expression of CD3, Foxp3, and IL-17, in combination with a DAPI counterstain. This assay allowed for the easy and clear identification of both tumor cells (using DAPI nuclear staining) and the infiltrating T-cell populations (using a combination of different markers). Following sample preparation and staining, a fluorescent microscope and imaging software were used to separate the different fluorescent colors used and to determine the number and type of cells present in both the tumor epithelium and the tumor-associated stroma.
An alternative assay to quantify and phenotype immune cell populations is flow cytometry analysis, or cytometry by time of flight (CyTOF) analysis, of tumor or peripheral samples (i.e., blood or ascites). Using this technique, all information about localization and relative distribution of the different cell types is lost. The use and analysis of peripheral samples also does not provide information about which cells are able to infiltrate the tumor microenvironment. Blood and ascite immune-cell analyses have been shown not to reflect the phenotype and frequency of immune cell infiltration of the tumor tissue8,9.
Another alternative is the use of bright-field microscopy. An advantage of this technique over fluorescence imaging is the absence of tissue autofluorescence. Although some samples contain more autofluorescence&#8212;particularly erythrocytes, but also other cell types, including neutrophilic granulocytes&#8212;these areas can easily be removed in the analysis of almost all samples. Immunofluorescence offers the advantage of analyzing multiple markers in one sample by using a panel of targeted fluorescent wavelengths. This is currently impossible to the same extent for bright-field microscopy due to the lack of a sufficient number of labels and commercially available antibody isotypes for a particular antigen.
The multicolor fluorescence IHC technique described here has been used in many different cancer types and antibody combinations to study different immune cell populations, as well as tumor cell-expressed molecules, such as human leukocyte antigens (HLA) and PD-L110,11,12. The protocol has been established and validated using many different types of samples and antibodies.

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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

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their relative distribution and their localization in the tissue. This technique has been extensively applied to study the immune infiltrate in various tumor types. The tumor microenvironment is infiltrated by immune cells that are attracted to the tumor site. Different immune cell populations have been found to play different roles in the tumor microenvironment and to have a different impact on the outcome of disease. This manuscript describes the use of multiparameter fluorescence IHC on oropharyngeal squamous cell carcinoma (OPSCC) as an example. This technique can be extended to other tissue samples and cell types of interest. In the presented study, we analyzed the intraepithelial and stromal compartment of a large OPSCC cohort (n = 162). We focused on total T lymphocytes (CD3+), immunosuppressive regulatory T cells (Tregs; i.e., FoxP3+), and T helper 17 (Th17) cells (i.e., IL-17+CD3+) using a nuclear counterstain to distinguish tumor epithelium from stroma. A high number of T cells was found to be correlated with improved disease-free survival in patients with a low number of intratumoral IL-17+ non-T cells. This suggests that IL-17+ non-T cells may be correlated with a poor immune response in OPSCC, which is in agreement with the correlation described between IL-17 and poor survival in cancer patients. Currently, novel multiparameter fluorescence IHC techniques are being developed using up to 7 different fluorochromes and will enable the more precise characterization and localization of immune cells in the tumor microenvironment.

A series of FFPE pretreatment tumor samples obtained from primary oropharyngeal tumors diagnosed in the Leiden University Medical Center, Leiden, the Netherlands between 1970 and 2011, selected as described before (n = 162), was stained using the protocol described13. One to four random images of each slide were analyzed (Figure 1). Some autofluorescent cells are indicated, which can clearly be distinguished by their complete yellow ap...

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

Multiparameter fluorescence immunohistochemistry can be used to assess the number, relative distribution, and localization of immune cell populations in the tumor microenvironment. This manuscript describes the use of this technique to analyze T-cell subpopulations in oropharyngeal cancer.

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

The overall goal of this experiment is to perform multicolor immunohistochemical visualization and enumeration of various T lymphocytes subpopulations in archival formalin-fixed paraffin-embedded tissue sections from oropharyngeal squamous cell carcinoma samples. This method can help answer key questions in immuno-oncology field such as, is there a link between tumor tissue infiltrating immune cells subpopulations and patient prognosis. With this technique, you can visualize several biomarkers in one tissue section.The implications of this technique extend towards cancer therapy, as the simultaneous evaluation of multiple immune cells or immune related factors might help in patient stratification for specific immune therapies. Begin by using a micro tongue to obtain four micrometer thick formalin-fixed paraffin-embedded tissue sections, laying the tissue ribbons on the surface of a 45 degree celsius de-ionized water bath as they are acquired. To capture the sections, place a glass slide in the water, directly underneath the tissue sample and carefully move the slide up at an angle.This section will stick to the glass surface as the slide is lifted out of the water. After drying the samples overnight at 37 degrees celsius, de-paraffinize the tissues with three five minute total immersions in 100%xylol. When the sections have been completed de-paraffinized, rehydrate the tissue samples with three descending ethanol immersions.Followed by a five minute wash in de-ionized water. Then, submerge the samples in boiling EDTA buffer for 10 minutes in the microwave and let the slides cool in the buffer for two hours. For labeling of the antigens of interest, rinse the samples with two five minute PBS washes.After the second wash, incubate the tissues with the primary antibodies and interest diluted in 1%bovine serum albumin in PBS at room temperature, overnight. The next morning, rinse the sections with three five minute PBS washes and incubate the samples with the appropriate secondary antibodies diluted in 1%serum in PBS at room temperature for one hour. At the end of the incubation, wash the slides three times in PBS as just demonstrated and mount the slides using an aqueous antifade mounting medium containing a nuclear counter stain.To analyze the samples, first select the 20 to 25 times objective on a fluorescence microscope and capture four images per slide using the appropriate filters for the secondary antibodies and counter stain. Save the images as they are obtained. Next, in the appropriate image analysis software, open the first image of interest and click on the overlay tab.Select the closed poly line shape to demarquee the boundary of the region of interest. When the shape is closed, the surface area of measurement will appear next to the region of interest. Save all the surface area data in the spreadsheet file to calculate the average surface areas and sound numbers per slide.Export the image as a TIFF or JPG file to be opened in ImageJ. Then, in ImageJ, open the first image of interest and select the cell counter under the plugins menu. Then, click initialize, click on type one and click on every cell designated as type one.Select a different type for each cell type to count the number of different types of cells in the image according to the fluorochrome expression. Finally, record the number of cells analyzed in each counter bin for each image in the spreadsheet file. In this representative experiment, on of four random images were analyzed from a series of formalin-fixed paraffin-embedded pretreatment tumor samples obtained from primary oropharyngeal tumors, diagnosed in the Leiden University Medical Center between 1970 to 2011, that were prepared as just demonstrated.In this experimental sample, some autofluorescent cells were observed, as distinguished by their complete yellow appearance, while negative control slides did not demonstrate any specific staining above background tissue autofluorescence. All of the tumor samples were determined to be infiltrated by varying numbers of fox p3 positive, CD3 positive T cells with only a small T cell subset expressing IL-17. Samples from patients with a high number of total infiltrating CD3 positive T cells demonstrate a trend toward a correlation with an improved disease-free survival compared to patient samples with low T cell members.Further, in samples with a low number of IL-17 positive cells, a high number of total infiltrating T cells is also correlated with an improved disease-free survival, suggesting that the effective tumor infiltrating T cells in oropharyngeal squamous cell carcinoma maybe related to the low number of IL-17 positive cells present. Once mastered, these sample preparation steps can be completed in one and a half days for a maximum of 25 slides per staining session. It's very important to de-paraffanize the sample, otherwise you'll get a lot of background.After its development, this technique paved the way for researchers in the fields of pathology and immunology. For exploring the microenvironment for the localisation of various immune cell subpopulations. After watching this video, you should have a good understanding of how to visualize various proteins and cell types in a single archival tissue section.Don't forget that working with silo is very dangerous, you should always work in a flow cabinet.

four-color-fluorescence-immunohistochemistry-t-cell-subpopulations

Research

JoVE Journal

Cancer Research

9.5K Views.  Leiden University Medical Center. The overall goal of this experiment is to perform multicolor immunohistochemical visualization and enumeration of various T lymphocytes subpopulations in archival formalin-fixed paraffin-embedded tissue sections from oropharyngeal squamous cell carcinoma samples. This method can help answer key questions in immuno-oncology field such as, is there a link between tumor tissue infiltrating immune cells subpopulations and patient prognosis. With this technique, you can visualize several biomarker...

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