This study was supported by Research Project&#160;of Shenzhen&#160;Health Family Planning System&#160;(grant#: SZBC2018001), the Natural Science Foundation of China (grant#: 81772754), and the Natural Science Foundation of Guangdong Province (grant#: 2017A030308009).

The Ethical Committee of the Seventh Affiliated Hospital, Sun Yat-sen University approved all experimental methods used in the study.
1. Preparation of the sample
<ol>
	<li>Immerse the fresh human tumor tissue (within 30 min after resection; cut into 2 cm x 2 cm x 0.3 cm) in 10% formalin for 24 h (at least 2 h). The volume of formalin should be at least ten times greater than that of the tissue.</li>
	<li>Set the program of the tissue processor as follows: 70% ethanol for 60 min, 80% ethanol...

<ol>
	<li>Galon, 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=Type,+density,+and+location+of+immune+cells+within+human+colorectal+tumors+predict+clinical+outcome.">Type, density, and location of immune cells within human colorectal tumors predict clinical outcome.</a> Science. 313 (5795), 1960-1964 (2006).</li><li>Hwang, W. T., Adams, S. F., Tahirovic, E., Hagemann, I. S., Coukos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+significance+of+tumor-infiltrating+T+cells+in+ovarian+cancer:+a+meta-analysis.">Prognostic significance of tumor-infiltrating T cells in ovarian cancer: a meta-analysis.</a> Gynecologic Oncology. 124 (2), 192-198 (2012).</li><li>Laghi, L., 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=CD3++cells+at+the+invasive+margin+of+deeply+invading+(pT3-T4)+colorectal+cancer+and+risk+of+post-surgical+metastasis:+a+longitudinal+study.">CD3+ cells at the invasive margin of deeply invading (pT3-T4) colorectal cancer and risk of post-surgical metastasis: a longitudinal study.</a> Lancet Oncology. 10 (9), 877-884 (2009).</li><li>Pages, F., 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=Effector+memory+T+cells,+early+metastasis,+and+survival+in+colorectal+cancer.">Effector memory T cells, early metastasis, and survival in colorectal cancer.</a> New England Journal of Medicine. 353 (25), 2654-2666 (2005).</li><li>Zhang, L., 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=Intratumoral+T+cells,+recurrence,+and+survival+in+epithelial+ovarian+cancer.">Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer.</a> New England Journal of Medicine. 348 (3), 203-213 (2003).</li><li>Sharma, P., 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=CD8+tumor-infiltrating+lymphocytes+are+predictive+of+survival+in+muscle-invasive+urothelial+carcinoma.">CD8 tumor-infiltrating lymphocytes are predictive of survival in muscle-invasive urothelial carcinoma.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (10), 3967-3972 (2007).</li><li>Mahmoud, S. 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=Tumor-infiltrating+CD8++lymphocytes+predict+clinical+outcome+in+breast+cancer.">Tumor-infiltrating CD8+ lymphocytes predict clinical outcome in breast cancer.</a> Journal of Clinical Oncology. 29 (15), 1949-1955 (2011).</li><li>Savas, P., 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=Single-cell+profiling+of+breast+cancer+T+cells+reveals+a+tissue-resident+memory+subset+associated+with+improved+prognosis.">Single-cell profiling of breast cancer T cells reveals a tissue-resident memory subset associated with improved prognosis.</a> Nature Medicine. 24 (7), 986-993 (2018).</li><li>Tumeh, P. C., 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=PD-1+blockade+induces+responses+by+inhibiting+adaptive+immune+resistance.">PD-1 blockade induces responses by inhibiting adaptive immune resistance.</a> Nature. 515 (7528), 568-571 (2014).</li><li>Kourea, H., Kotoula, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+tumor+immunodiagnostics.">Towards tumor immunodiagnostics.</a> Annals of Translational Medicine. 4 (14), 263 (2016).</li><li>Smith, N. R., Womack, 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+matrix+approach+to+guide+IHC-based+tissue+biomarker+development+in+oncology+drug+discovery.">A matrix approach to guide IHC-based tissue biomarker development in oncology drug discovery.</a> Journal of Pathology. 232 (2), 190-198 (2014).</li></ol>

The presence of CD8+ TILs has been reported in different kind of tumors6,7,8. The retrieval of the antigen denatures the CD8 protein and exposes the antigen epitope. Binding of the anti-CD8 antibody and subsequent horseradish peroxidase-labeled secondary antibody are quite effective and the color development is well established. The results of CD8 IHC can help to provide a direct view of CD8+ TILs in the tumor microenvironment, ...

The presence of tumor-infiltrating lymphocytes (TILs) is considered to be associated with better clinical outcomes in different kinds of cancer1,2,3,4,5. T-cell surface glycoprotein CD8 (CD8)+ TILs are the most important effectors to prevent tumor development among all TILs6,7,8. The application of IHC can be used to help researchers and/or pathologists accurately observe the CD8+ TILs of individual patients. Assessment of CD8+ TILs can help to determine the prognosis for patients. In addition, CD8+ TIL evaluation can be one of the indicators for tumor immunotherapy9.
IHC is the most commonly used detection method to evaluate specific proteins in patients&#8217; organs or tissues, especially human tumor tissues, both qualitatively and quantitatively10,11. IHC is widely used in pathological diagnosis. Specimen collection and storage for IHC are relatively easy. For example, there is little time limitation on the use of a sample as long as tissues are infiltrated with paraffin wax. The forms of antigen are well preserved. Thus, it is possible to handle many tissue samples at the same time. The experimental conditions can be controlled to avoid human interference. However, there are still some limitations of IHC in terms of sensitivity and background reduction.

<table border="0" cellpadding="0" cellspacing="0" style="width:908px;" width="908">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:352px;">Ammonium hydroxide</td>
			<td style="width:189px;">Guangzhou Chemical Reagent Factory</td>
			<td style="width:119px;">1336-21-6</td>
			<td style="width:248px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Antibody Diluent, Background Reducing</td>
			<td style="width:189px;">DAKO</td>
			<td style="width:119px;">S302281</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Blocking buffer&#160;</td>
			<td style="width:189px;">Serotec</td>
			<td style="width:119px;">BUF029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">CD8, Rabbit Monoclonal Antibody</td>
			<td style="width:189px;">Thermo Scientific</td>
			<td style="width:119px;">RM-9116-S&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Citrate buffer</td>
			<td style="width:189px;">MXB Biotechnologies</td>
			<td style="width:119px;">MVS-0066</td>
			<td style="width:248px;">Antigen retrieval buffer, pH 6.0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">DAB</td>
			<td style="width:189px;">MXB Biotechnologies</td>
			<td style="width:119px;">DAB-0031</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:352px;">Ethanol</td>
			<td style="width:189px;">Guangzhou Chemical Reagent Factory</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Formalin</td>
			<td style="width:189px;">Xiuwei Commerce</td>
			<td style="width:119px;">XW-RS-019</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:352px;">Goat anti-Rabbit IgG (H+L)&#160;Cross Adsorbed Secondary Antibody, HRP conjugate&#160;</td>
			<td style="width:189px;">Thermo Scientific</td>
			<td style="width:119px;">31462</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Hematoxylin</td>
			<td style="width:189px;">Xiuwei Commerce</td>
			<td style="width:119px;">XW-RS-001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:352px;">Hydrochloric</td>
			<td style="width:189px;">Guangzhou Chemical Reagent Factory</td>
			<td style="width:119px;">7647-01-0</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:352px;">Hydrogen peroxide</td>
			<td style="width:189px;">Guangzhou Chemical Reagent Factory</td>
			<td style="width:119px;">7722-84-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Paraffin</td>
			<td style="width:189px;">Leica</td>
			<td style="width:119px;">39601006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Phosphate-buffered saline(PBS)&#160;</td>
			<td style="width:189px;">MXB Biotechnologies</td>
			<td style="width:119px;">PBS-0060</td>
			<td>pH 7.5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Pressure cooker</td>
			<td style="width:189px;">Supor</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Resin</td>
			<td style="width:189px;">Xiuwei Commerce</td>
			<td style="width:119px;">XW-RS-005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Tissue processor</td>
			<td style="width:189px;">Leica&#160;</td>
			<td style="width:119px;">TP1020</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:352px;">Xylene</td>
			<td style="width:189px;">Guangzhou Chemical Reagent Factory</td>
			<td style="width:119px;">1330-20-7</td>
			<td></td>
		</tr>
	</tbody>
</table>

immunohistochemical staining of cd8 protein in tissue sections from patients with tumors

Immunohistochemistry (IHC) is one of the most useful detection techniques in scientific research and clinical practice. IHC can give researchers a direct view of a target protein or target cells through bi-colored images of histological tissue sections from patients. Cell nuclei are stained with hematoxylin and target proteins are stained via the chromogenic reaction of 3,3&#39;,4,4&#39;-Biphenyltetramine tetrahydrochloride (DAB) in classic IHC, which can show both the relative expression level of a target protein and its location within the tissue. The principle utilized in IHC is the specific binding between an antigen and an antibody, which partially guarantees the accuracy of the results. IHC is also widely used to study cell subsets because it can show the exact location of target cell subsets in organs or tissues. This can help us understand their effects and functional mechanisms. Clinical data suggest that T-cell surface glycoprotein CD8 (CD8)+ tumor-infiltrating lymphocytes (TILs) could serve as an indication of the effectiveness of anti-programmed cell death 1 (PD-1) / programmed cell death ligand 1 (PD-L1) therapy in patients with tumors; therefore, IHC staining of CD8 protein to evaluate the CD8+ TILs in tissue sections becomes very important. IHC has several advantages. Samples are more accessible and last for a long time in storage. The reagents and equipment have been commercialized for years. However, there are also limitations. Lymphocyte infiltration into tumors is a dynamic process, and the results of IHC only reflect the infiltration at one specific time point, and not the dynamic changes over time. This disadvantage partly inhibits its clinical application in tumor immunotherapy, which mostly depends on T cell infiltration into the tumor microenvironment.

Successful immunohistochemical staining shows the CD8+ TILs in the tumor sections (bladder cancer). CD8 protein expression (the surface marker of CD8+ TILs) are defined and quantified using the brown signal in the image (Figure 1); the blue signal represents the cell nucleus. Meanwhile, the location of CD8+ TILs can be determined by observing the distribution of both the brown and blue signals (Figure 2). Figure 3 and Figure 4</st...

The present study presents a protocol for immunohistochemistry (IHC) that can image specific antigens located on tissue sections and determine the relative expression level of a target protein or the distribution of target cells.

Immunohistochemical Staining of CD8 Protein in Tissue Sections from Patients with Tumors

Immunohistochemistry (IHC) is one of the most useful detection techniques in scientific research and clinical practice. IHC can give researchers a direct ...

Immunohistochemical Staining of CD8 Protein in Tissue Sections from Patients with Tumors

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