This work was supported by the National Natural Science Foundation of China (NO.81860413, 81960455), Yunnan Science and Technology Department Fund (202001AY070001-080), Scientific Research Foundation of Education Department of Yunnan Province(2019J1274).

The research was approved by the medical ethics committee of Yunnan Cancer Hospital/the Third Affiliated Hospital of Kunming Medical University. All the subjects/legal guardians signed informed consent.
1. Slide preparation
<ol>
	<li>Cut sections of paired paraffin blocks containing primary lung tumor or lung cancer brain metastases cells at a thickness of 4 &#181;m using a microtome. Remove sections to water and separate with tweezers, choose the best one and adhere it onto the polylysine-coated slide.</li>
	<li>Place the slides of tissues in an oven at 65 &#176;C for 30 min to enhance tissue adhesion.</li>
	<li>Immerse the slides in xylene through three changes, each lasting for 10 min.</li>
	<li>Gradually reduce the alcohol concentration and incubate slides in 100% ethanol for 5 min, 90% ethanol for 5 min, 75% ethanol for 5 min, and in deionized water for 3 min.</li>
</ol>
2. Heat-induced epitope retrieval (HIER)
<ol>
	<li>Dilute 100x sodium citrate buffer solution (pH 6.0) to 1x (10 mM) in deionized water, preparing enough buffer solution to completely submerge the slides.</li>
	<li>Place the slides in a pressure cooker, subjecting them to high heat (100 &#176;C) and pressure (~30 psi) for 2 min. After heating, allow the slides to cool to room temperature in distilled water for 3 min.</li>
	<li>Dissolve one packet of 52 g of phosphate-buffered saline (PBS; powdered) in 5 L of deionized water for preparing PBS buffer solution (pH 7.0). Place the slides in PBS buffer solution for 5 min, with 3 changes.</li>
</ol>
3. Peroxidase blocking
<ol>
	<li>Cover the sections with 3% hydrogen peroxide and incubate for 10 min at room temperature. Rinse the slides in PBS, 3x for 5 min each.</li>
	<li>Use filter paper to absorb excess water away from the perimeter of the tissue. Meanwhile, make sure that the tissue is moist.</li>
</ol>
4. Primary antibody incubation for first round
<ol>
	<li>Prepare a working mixture of the primary antibodies for CD8 (Rabbit monoclonal antibody, clone SP16) and CD20 (mouse monoclonal antibody, clone L26), diluted 1:50 in Bond primary antibody diluent.</li>
	<li>Add the antibody complex onto the sections and incubate for 1 h at room temperature.</li>
	<li>Prepare a solution of 0.1% Tween/phosphate-buffered saline: 1 mL of Tween in 1 L of PBS buffer solution.</li>
	<li>Wash the sections with 0.1% Tween/phosphate-buffered saline, 3x for 5 min each. Use filter paper to draw excess water away from the perimeter of the tissue, meanwhile, make sure the tissue is moist.</li>
</ol>
5. Secondary antibody incubation for first round
<ol>
	<li>Prepare a mixture of fluorescence-labeled goat anti-rabbit antibody (Excitation (Ex): 495 nm) and goat anti-mouse antibody (Ex: 578 nm), diluted 1:50 in phosphate buffer saline. The goat anti-rabbit antibody attaches to the primary antibody of CD8, and the goat anti-mouse antibody attaches to the primary antibody of CD20.</li>
	<li>Add the secondary antibody mixture dropwise and incubate at room temperature for 1 h.</li>
</ol>
6. Heat-induced epitope retrieval and peroxidase blocking
<ol>
	<li>Dilute 100x sodium citrate (pH 6.0) to 1x (10 mM) in deionized water, preparing enough buffer solution to completely submerge the slides.</li>
	<li>Place the slides in a pressure cooker and subject them to high heat (100 &#176;C) and pressure (~30 psi) for 1 min. After heating, place the slides in distilled water to cool to room temperature for 3 min.</li>
	<li>Perform peroxidase blocking as described in step 3.</li>
</ol>
7. Primary antibody incubation for second round
<ol>
	<li>Prepare a working mixture of the primary antibodies for CD3 (Rabbit monoclonal antibody, clone SP7) and CK (mouse monoclonal antibody, MX005), diluted 1:50 in primary antibody diluent.</li>
	<li>Add the antibody complex onto the sections and incubate for 1 h at room temperature.</li>
	<li>Wash with 0.1% Tween/phosphate-buffered saline, 3x for 5 min each. Use filter paper to draw excess water away from the perimeter of the tissue, meanwhile, make sure the tissue is moist.</li>
</ol>
8. Secondary antibody incubation for second round
<ol>
	<li>Prepare goat anti-rabbit (Ex: 652 nm) and goat anti-mouse antibody (Ex: 590 nm) mixture, dilution 1:50 in phosphate buffer saline. The goat anti-rabbit antibody attaches to the primary antibody of CD3, and the goat anti-mouse antibody attaches to the primary antibody of CK.</li>
	<li>Add secondary antibody mixture dropwise, incubate at room temperature for 1 h. Wash with 0.1% Tween/phosphate-buffered saline, 3x for 5 min each.</li>
</ol>
9. Autofluorescence quenching and DAPI staining
<ol>
	<li>Add reagent (0.15 M/L KMnO4) to the tissue section for 1 min. Rinse with running water for 5 min. 
	CAUTION: KMnO4 is toxic and damages the skin. Make sure to wear gloves when handling the slides. If the liquid drips onto the skin, wipe it off quickly with clean napkins and flush with flowing water.</li>
	<li>Dry the slide with increasing concentrations of alcohol (70%, 90%, 100%), for 3 min in each concentration.</li>
	<li>Add DAPI and coverslip for multispectral imaging. The amount of DAPI depends on the tissue size. Make sure the tissue is completely covered by DAPI after the coverslip is added.</li>
</ol>
10. Slide scanning
<ol>
	<li>Place the slides on a tray and push until it cannot be pushed further.</li>
	<li>To start the program, double-click on the Program Icon on the desktop. During start-up, the mode selection window is displayed. The selection window displays two brightfield and fluorescent modes: automatic mode and manual mode. In fluorescent scan mode, click Automatic Mode.</li>
	<li>Move the mouse cursor over ? button to display the information about the settings. Click Filter Setting &#62; Filter Channel to choose the Channel Number &#62; Pseudo Color. Define the color and save.</li>
	<li>Click Routine Work &#62; Scanning Mode &#62; Full Automatic. Click Routine Work &#62; Slide Name to define slide name for output. Click Routine Work &#62; Channel Settings to choose filters.</li>
	<li>Click Routine Work &#62; Scan Options to determine the resulting quality and storage location of the virtual slide.</li>
	<li>Click Preview for threshold setting and selection of the range to be scanned.</li>
	<li>Click Hardware &#62; Filters &#62; Live &#62; Auto Focus &#62; Auto Exposure &#62; Digital Gain &#62; Tick use manual exposure time &#62; Tick limiting the range &#62; Set Current.</li>
	<li>Click Routine Work &#62; Start Scan. Select magnification level as 20x or 40x. Choose 20x for appropriate file sizes. MRXS extension is defined by 3D Pannoramic MIDI scanner. Image extension can also be changed to TIFF image.</li>
	<li>Click Slide Viewer &#62; Multiview Toolbox to choose the image for confocal, then align and integrate the image.</li>
</ol>
11. Quantitative evaluation of cell densities
<ol>
	<li>Quantify percentages of positive immune cells (CD3+, CD3+CD8+, CD20+) in tumor and stroma areas with Halo 10 scanner software. Validate CK staining to define tumor tissue.</li>
</ol>

In Situ Immunohistochemical Detection of Immune Cell Markers in Cancer Tissues

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+imaging+in+early+stage+non-small+cell+lung+cancer:+still+a+controversial+topic.">Brain imaging in early stage non-small cell lung cancer: still a controversial topic.</a> J Thorac Dis. 10, S2168-S2171 (2018).</li><li>Vilari&#241;o, N., Bruna, J., Bosch-Barrera, J., Valiente, M., Nadal, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunotherapy+in+NSCLC+patients+with+brain+metastases.+Understanding+brain+tumor+microenvironment+and+dissecting+outcomes+from+immune+checkpoint+blockade+in+the+clinic.">Immunotherapy in NSCLC patients with brain metastases. Understanding brain tumor microenvironment and dissecting outcomes from immune checkpoint blockade in the clinic.</a> Cancer Treat Rev. 89, 102067 (2020).</li><li>Babar, Q., Saeed, A., Tabish, T. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry+Innovations+for+Diagnosis+and+Tissue-Based+Biomarker+Detection.">Immunohistochemistry Innovations for Diagnosis and Tissue-Based Biomarker Detection.</a> Curr Hematol Malig Rep. 14 (5), 368-375 (2019).</li><li>Janardhan, K. S., Jensen, H., Clayton, N. P., Herbert, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry+in+Investigative+and+Toxicologic+Pathology.">Immunohistochemistry in Investigative and Toxicologic Pathology.</a> Toxicol Pathol. 46 (5), 488-510 (2018).</li><li>Torlakovic, E. E., Nielsen, S., Vyberg, M., Taylor, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+controls+under+control:+the+time+is+now+for+immunohistochemistry.">Getting controls under control: the time is now for immunohistochemistry.</a> J Clin Pathol. 68 (11), 879-882 (2015).</li><li>Tan, W. C. 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=Overview+of+multiplex+immunohistochemistry/immunofluorescence+techniques+in+the+era+of+cancer+immunotherapy.">Overview of multiplex immunohistochemistry/immunofluorescence techniques in the era of cancer immunotherapy.</a> Cancer Commun (Lond). 40 (4), 135-153 (2020).</li><li>Wong, P. 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=Multiplex+quantitative+analysis+of+tumor-infiltrating+lymphocytes+and+immunotherapy+outcome+in+metastatic+melanoma.">Multiplex quantitative analysis of tumor-infiltrating lymphocytes and immunotherapy outcome in metastatic melanoma.</a> Clin Cancer Res. 25 (8), 2442-2449 (2019).</li><li>Sanchez, K., 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=Multiplex+immunofluorescence+to+measure+dynamic+changes+in+tumor-infiltrating+lymphocytes+and+PD-L1+in+early-stage+breast+cancer.">Multiplex immunofluorescence to measure dynamic changes in tumor-infiltrating lymphocytes and PD-L1 in early-stage breast cancer.</a> Breast Cancer Res. 23 (1), 2 (2021).</li><li>Zhang, W., 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=Multiplex+immunohistochemistry+indicates+biomarkers+in+colorectal+cancer.">Multiplex immunohistochemistry indicates biomarkers in colorectal cancer.</a> Neoplasma. 68 (6), 1272-1282 (2021).</li><li>Salameh, S., Nouel, D., Flores, C., Hoops, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+immunohistochemistry+protocol+for+detecting+the+guidance+cue+Netrin-1+in+neural+tissue.">An optimized immunohistochemistry protocol for detecting the guidance cue Netrin-1 in neural tissue.</a> MethodsX. 5, 1-7 (2018).</li><li>McClellan, P., Jacquet, R., Yu, Q., Landis, W. 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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=Sudan+Black+B+treatment+reduces+autofluorescence+and+improves+resolution+of+in+situ+hybridization+specific+fluorescent+signals+of+brain+sections.">Sudan Black B treatment reduces autofluorescence and improves resolution of in situ hybridization specific fluorescent signals of brain sections.</a> Histol Histopathol. 25 (8), 1017-1024 (2010).</li><li>Ahrens, M. J., Dudley, A. 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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

We have described the process for multiplex cyclic fluorescence immunohistochemistry staining. The primary antibody selection is a crucial aspect of the fluorescence immunohistochemistry assay, and monoclonal antibodies are recommended for better specificity and repeatability. To optimize the working concentration of the primary antibody, a series of dilutions have been tested through immunohistochemistry experiments. Both positive controls (to assess target antigen expression) and negative controls (no primary antibody ...

Brain metastases (BM) represent the most common central nervous system (CNS) tumors, occurring in nearly half of non-small cell lung cancer cases (NSCLC), with a poor prognosis1. An estimated 10%-20% of NSCLC patients already have BM at the time of initial diagnosis, and approximately 40% of NSCLC cases will develop BM during the course of treatment2. The tumor microenvironment (TME) is closely associated with NSCLC occurrence and BM, including various components, such as blood vessels, fibroblasts, macrophages, extracellular matrix (ECM), lymphoid, bone marrow-derived immune cells, and signaling molecules3,4. Microenvironmental immune cells play a crucial role in influencing cancer cell growth and development. Brain metastases present numerous potential treatment targets characterized by complex immunological microenvironments and signaling processes. For instance, PD-1 inhibitors have shown clinical efficacy for patients with lung cancer brain metastasis (LCBM) as an immune-checkpoint inhibitor (ICI). However, the frequency of responses to PD-1 therapy varies between primary NSCLC and LCBM5, suggesting that the tumor immune microenvironment acts as a critical ICI regulator.
Immunohistochemistry (IHC) is an invaluable tool in the fields of biology, foundation medicine, and pathology6. This detection method visualizes antigen expression through the interaction of antigen-antibody on a tissue slide7. IHC is used for diagnosing predictive markers, evaluating prognostic markers, guiding targeted therapies, and exploring the biological functions of tumor cells8. However, the traditional IHC method can only detect one biomarker at a time. To address this limitation, the innovation of immunohistochemical technology has led to the development of multiplex fluorescence immunohistochemistry (mfIHC), which allows for the simultaneous identification of multiple protein markers on the same tissue slide, both in bright field and fluorescent field9. This advancement provides accurate analysis of cell composition and molecular interactions among stromal cells, immune cells, and cancer cells within the TME.
In this study, we present a protocol for multiplex cyclic immunohistochemistry to analyze the spatial distribution of immune cells. Two primary antibodies of different species, such as rabbit and rat, are chosen for incubation simultaneously, followed by fluorescence-labeled secondary antibodies. Antigen retrieval is performed after each round of antigen-antibody reaction. Autofluorescence is blocked, and 4', 6-diamidino-2-phenylindole (DAPI) is used for staining the nuclei. The panel includes sequential detection of CD3, CD8, CD20, and CK, cells are categorized according to the markers: tumor cells (CK+), mature T cells (CD3+), cytotoxic T cells (CD3+CD8+), B cells (CD20+)10,11.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.15 mol/L KmnO4</td>
			<td>Maixin Biotechnology Co. Ltd.</td>
			<td>MST-8005</td>
			<td></td>
		</tr>
		<tr>
			<td>100x sodium citrate&#160;</td>
			<td>Maixin Biotechnology Co., Ltd</td>
			<td>MVS-0100</td>
			<td></td>
		</tr>
		<tr>
			<td>3% hydrogen peroxide</td>
			<td>Maixin Biotechnology Co., Ltd</td>
			<td>SP KIT-A1</td>
			<td></td>
		</tr>
		<tr>
			<td>3D Pannoramic MIDI</td>
			<td>3D histech Ltd</td>
			<td>Pannoramic MIDI 1.18</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488</td>
			<td>Abcam</td>
			<td>ab150113</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 568&#160;</td>
			<td>Abcam</td>
			<td>ab175701</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594</td>
			<td>Abcam</td>
			<td>ab150116</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 647</td>
			<td>Abcam</td>
			<td>ab150079</td>
			<td></td>
		</tr>
		<tr>
			<td>Bond primary antibody diluent</td>
			<td>Lecia</td>
			<td>AR9352</td>
			<td></td>
		</tr>
		<tr>
			<td>CD20</td>
			<td>Maixin Biotechnology Co., Ltd</td>
			<td>kit-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3</td>
			<td>Maixin Biotechnology Co., Ltd.</td>
			<td>&#160;kit-0003</td>
			<td></td>
		</tr>
		<tr>
			<td>CD8&#160;</td>
			<td>Maixin Biotechnology Co., Ltd</td>
			<td>RMA-0514</td>
			<td></td>
		</tr>
		<tr>
			<td>CK</td>
			<td>Maixin Biotechnology Co. Ltd.</td>
			<td>MAB-0671,</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>sig-ma</td>
			<td>D8417</td>
			<td></td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>Sinopharm Group Chemical reagent Co., LTD</td>
			<td>10009218</td>
			<td></td>
		</tr>
		<tr>
			<td>Histocore Multicut</td>
			<td>lecia</td>
			<td>2245</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS(powder)</td>
			<td>Maixin Biotechnology Co., Ltd</td>
			<td>PBS-0061</td>
			<td></td>
		</tr>
		<tr>
			<td>slide viwer&#160;</td>
			<td>3D histech Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>xylene</td>
			<td>Sinopharm Group Chemical reagent Co., LTD</td>
			<td>10023418</td>
			<td></td>
		</tr>
	</tbody>
</table>

multiplex cyclic fluorescent immunohistochemistry

The tumor microenvironment involves interactions between host cells, tumor cells, immune cells, stromal cells, and vasculature. Characterizing and spatially organizing immune cell subsets and target proteins are crucial for prognostic and therapeutic purposes. This has led to the development of multiplexed immunohistochemistry staining methods. Multiplex fluorescence immunohistochemistry allows the simultaneous detection of multiple markers, facilitating a comprehensive understanding of cell function and intercellular interactions. In this paper, we describe a workflow for the multiplex cyclic fluorescent immunohistochemistry assay and its application in the quantification analysis of lymphocyte subsets. The multiplex cyclic fluorescent immunohistochemistry staining follows similar steps and reagents as standard immunohistochemistry, involving antigen retrieval, cyclic antibody incubation, and staining on a formalin-fixed paraffin-embedded (FFPE) tissue slide. During the antigen-antibody reaction, a mixture of antibodies from different species is prepared. Conditions, such as antigen retrieval time and antibody concentration, are optimized and validated to increase the signal-to-noise ratio. This technique is reproducible and serves as a valuable tool for immunotherapy research and clinical applications.

Author Spotlight: Efficient Detection of Immune Cell-Infiltration in Cancer Tissues Using Fluorescent Immunohistochemistry

Multiplex Cyclic Fluorescent Immunohistochemistry

The challenge for immunohistochemistry is to choose the species of primary and secondary monoclonal antibodies to avoid cross-reactivity and obtain better specificity and repeatability. Gender decision is necessary to optimize the antibody dilution ratio. Meanwhile, another challenge is to align the localization of and the sub structures.The use of this protocol meet the technical evolution of the distribution of three lymphocytic subpopulations, such as CT three, positive CT three, and CT eight positive and CT 20 positive based on in cell counts and the protein expression in conserv tissues, the level of infiltration can be observed too. In our protocol, the parameter Antibodies and the Fluor secondary antibodies derive from different species at the iso mixture making us a efficient. This measure also shows a specific measure between antibody and antigen.

We present a protocol for cyclic antigen detection using 5-color multiplex fluorescence on a single slide. Through our optimization of the assay, we enable the incubation of two antibodies from different species (Figure 1). The necessary devices for the experiment procedure include a pressure cooker and immunostaining box (Figure 2A).
After completing the assay, we define pseudo color of the four markers before scanning the slides. Th...

Watch this Scientific Journal Video about Author Spotlight: Efficient Detection of Immune Cell-Infiltration in Cancer Tissues Using Fluorescent Immunohistochemistry at JoVE.com

Multiplex cyclic immunohistochemistry allows in situ detection of multiple markers simultaneously using repeated antigen-antibody incubation, image scanning, and image alignment and integration. Here, we present the operating protocol for identifying immune cell substrates with this technology in lung cancer and paired brain metastasis samples.

The tumor microenvironment involves interactions between host cells, tumor cells, immune cells, stromal cells, and vasculature. Characterizing and ...

multiplex-cyclic-fluorescent-immunohistochemistry

Research

JoVE Journal

Cancer Research

Preparation of Slides for Multiplex Cyclic Immunohistochemistry of Cancer Tissue Sections

Multiplex Cyclic Fluorescent Immunohistochemical Staining of Cancer Tissue Sections

447 Views.  The Third Affiliated Hospital of Kunming Medical University & Yunnan Cancer Hospital.  Multiplex cyclic immunohistochemistry allows in situ detection of multiple markers simultaneously using repeated antigen-antibody incubation, image scanning, and image alignment and integration. Here, we present the operating protocol for identifying immune cell substrates with this technology in lung cancer and paired brain metastasis samples.

In Situ Immunohistochemical Detection of Immune Cell Markers in Cancer Tissues

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