Name: multiplex cyclic fluorescent immunohistochemistry
Uploaded: 2024-01-26T14:35:00.000+00:00
Duration: 4 min 21 s
Description: Watch this Scientific Journal Video about Author Spotlight: Efficient Detection of Immune Cell-Infiltration in Cancer Tissues Using Fluorescent Immunohistochemistry at JoVE.com

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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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><tbody><tr><td>0.15 mol/L KmnO&lt;sub&gt;4&lt;/sub&gt;</td><td>Maixin Biotechnology Co. Ltd.</td><td>MST-8005</td><td></td></tr><tr><td>100x sodium citrate&amp;nbsp;</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&amp;nbsp;</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>&amp;nbsp;kit-0003</td><td></td></tr><tr><td>CD8&amp;nbsp;</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&amp;nbsp;</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.

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

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

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.

Multiplex Cyclic Fluorescent Immunohistochemistry

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

632 Views.  The Third Affiliated Hospital of Kunming Medical University & Yunnan Cancer Hospital. De uitdaging voor immunohistochemie is om de soorten primaire en secundaire monoklonale antilichamen te kiezen om kruisreactiviteit te voorkomen en een betere specificiteit en herhaalbaarheid te verkrijgen. Geslachtsbepaling is noodzakelijk om de verdunningsverhouding van antilichamen te optimaliseren. Ondertussen is een andere uitdaging het op elkaar afstemmen van de lokalisatie van en de substructuren.Het gebruik van dit protocol voldoet aan de technische evolutie van de verdeling va...

Video: Auteur in de schijnwerpers: Efficiënte detectie van infiltratie van immuuncellen in kankerweefsels met behulp van fluorescerende immunohistochemie

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here.

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens

Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of tissue samples from formalin-fixed, paraffin-embedded (FFPE) biopsies has become a key tool for understanding the complexity of tumor immunology and discovering novel predictive biomarkers for cancer immunotherapy. Immunoprofiling analysis of tissues requires the evaluation of combined markers, including inflammatory cell subpopulations and immune checkpoints, in the tumor microenvironment. The advent of novel multiplex immunohistochemical methods allows for a more efficient multiparametric analysis of single tissue sections than does standard monoplex immunohistochemistry (IHC). One commercially available multiplex immunofluorescence (IF) method is based on tyramide-signal amplification and, combined with multispectral microscopic analysis, allows for a better signal separation of diverse markers in tissue. This methodology is compatible with the use of unconjugated primary antibodies that have been optimized for standard IHC on FFPE tissue samples. Herein we describe in detail an automated protocol that allows multiplex IF labeling of carcinoma tissue samples with a six-marker multiplex antibody panel comprising PD-L1, PD-1, CD68, CD8, Ki-67, and AE1/AE3 cytokeratins with 4&#8242;,6-diamidino-2-phenylindole as a nuclear cell counterstain. The multiplex panel protocol is optimized in an automated IHC stainer for a staining time that is shorter than that of the manual protocol and can be directly applied and adapted by any laboratory investigator for immuno-oncology studies on human FFPE tissue samples. Also described are several controls and tools, including a drop-control method for fine quality control of a new multiplex IF panel, that are useful for the optimization and validation of the technique.

A detailed protocol for a six-marker multiplex immunofluorescence panel is optimized and performed, using an automated stainer for more consistent results and a shorter procedure time. This approach can be directly adapted by any laboratory for immuno-oncology studies.

Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of ...

Optimization, Design and Avoiding Pitfalls in Manual Multiplex Fluorescent Immunohistochemistry

Microenvironment evaluation of intact tissue for analysis of cell infiltration and spatial organization are essential in understanding the complexity of disease processes. The principle techniques used in the past include immunohistochemistry (IHC) and immunofluorescence (IF) which enable visualization of cells as a snapshot in time using between 1 and 4 markers. Both techniques have shortcomings including difficulty staining poorly antigenic targets and limitations related to cross-species reactivity. IHC is reliable and reproducible, but the nature of the chemistry and reliance on the visible light spectrum allows for only a few markers to be used and makes co-localization challenging. Use of IF broadens potential markers but typically relies on frozen tissue due to the extensive tissue autofluorescence following formalin fixation. Flow cytometry, a technique that enables simultaneous labeling of multiple epitopes, abrogates many of the deficiencies of IF and IHC, however, the need to examine cells as a single cell suspension loses the spatial context of cells discarding important biologic relationships. Multiplex fluorescent immunohistochemistry (mfIHC) bridges these technologies allowing for multi-epitope cellular phenotyping in formalin fixed paraffin embedded (FFPE) tissue while preserving the overall microenvironment architecture and spatial relationship of cells within intact undisrupted tissue. High fluorescent intensity fluorophores that covalently bond to the tissue epitope enables multiple applications of primary antibodies without worry of species specific cross-reactivity by secondary antibodies. Although this technology has been proven to produce reliable and accurate images for the study of disease, the process of creating a useful mfIHC staining strategy can be time consuming and exacting due to extensive optimization and design. In order to make robust images that represent accurate cellular interactions in-situ and to mitigate the optimization period for manual analysis, presented here are methods for slide preparation, optimizing antibodies, multiplex design as well as errors commonly encountered during the staining process.

Multiplex fluorescent immunohistochemistry is an emerging technology that enables the visualization of multiple cell types within intact formalin-fixed, paraffin embedded (FFPE) tissue. Presented are guidelines for ensuring a successful 7-color multiplex with instructions for optimizing antibodies and reagents, preparing slides, design and tips for avoiding common problems.

Microenvironment evaluation of intact tissue for analysis of cell infiltration and spatial organization are essential in understanding the complexity of ...

Multiplex Immunofluorescence and Spatial Image Analysis of the Tumor Microenvironment

Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment

The tumor microenvironment (TME) is composed of a plethora of different cell types, such as cytotoxic immune cells and immunomodulatory cells. Depending on its composition and the interactions between cancer cells and peri-tumoral cells, the TME may affect cancer progression. The characterization of tumors and their complex microenvironment could improve the understanding of cancer diseases and may help scientists and clinicians to discover new biomarkers.
We recently developed several multiplex immunofluorescence (mIF) panels based on tyramide signal amplification (TSA) for the characterization of the TME in colorectal cancer, head and neck squamous cell carcinoma, melanoma, and lung cancer. Once the staining and scanning of the corresponding panels are completed, the samples are analyzed on an image analysis software. The spatial position and the staining of each cell are then exported from this quantification software into R. We developed R scripts that allow us not only to analyze the density of each cell type in several tumor compartments (e.g. the center of the tumor, the margin of the tumor, and the stroma) but also to perform distance-based analyses between different cell types.
This particular workflow adds a spatial dimension to the classical density analysis already routinely performed for several markers. mIF analysis could allow scientists to have a better understanding of the complex interaction between cancer cells and the TME and to discover new predictive biomarkers of response to treatments, such as immune checkpoint inhibitors, and targeted therapies.

Author Spotlight: Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment  

In this article, a protocol for manual tyramide signal amplification (TSA) multiplex immunofluorescence (mIF) combined with image analysis and spatial analysis is described. This protocol can be used with formalin-fixed paraffin-embedded (FFPE) sections for the staining of two to six antigens per slide depending on the slide scanner available in the laboratory.

The tumor microenvironment (TME) is composed of a plethora of different cell types, such as cytotoxic immune cells and immunomodulatory cells. Depending ...

Mapping the Tumor Immune Landscape: An In-Depth Multiplex IHC Analysis Protocol

Multiplex Immunohistochemical Analysis of the Spatial Immune Cell Landscape of the Tumor Microenvironment

The immune cell landscape of the tumor microenvironment potentially contains information for the discovery of prognostic and predictive biomarkers. Multiplex immunohistochemistry is a valuable tool to visualize and identify different types of immune cells in tumor tissues while retaining its spatial information. Here we provide detailed protocols to analyze lymphocyte, myeloid, and dendritic cell populations in tissue sections. Starting from cutting formalin-fixed paraffin-embedded sections, automatic multiplex staining procedures on an automated platform, scanning of the slides on a multispectral imaging microscope, to the analysis of images using an in-house-developed machine learning algorithm ImmuNet. These protocols can be applied to a variety of tumor specimens by simply switching tumor markers to analyze immune cells in different compartments of the sample (tumor versus invasive margin) and apply nearest-neighbor analysis. This analysis is not limited to tumor samples but can also be applied to other (non-)pathogenic tissues. Improvements to the equipment and workflow over the past few years have significantly shortened throughput times, which facilitates the future application of this procedure in the diagnostic setting.

Author Spotlight: Unlocking Insights into the Immune Cell Landscape of Tumors

This protocol describes in detail how immune cell characterization of the tumor microenvironment using multiplex immunohistochemistry is carried out.

The immune cell landscape of the tumor microenvironment potentially contains information for the discovery of prognostic and predictive biomarkers. ...

Deciphering Tumor Microenvironment Using mIHC in Lung Squamous Carcinoma

Multiplex Immunohistochemistry Staining for Paraffin-embedded Lung Cancer Tissue

Lung cancer is the leading cause of malignant tumor-related morbidity and mortality all over the world, and the complex tumor microenvironment has been considered the leading cause of death in lung cancer patients. The complexity of the tumor microenvironment requires effective methods to understand cell-to-cell relationships in tumor tissues. The multiplex immunohistochemistry (mIHC) technique has become a key tool for inferring the relationship between the expression of proteins upstream and downstream of signaling pathways in tumor tissues and developing clinical diagnoses and treatment plans. mIHC is a multi-label immunofluorescence staining method based on Tyramine Signal Amplification (TSA) technology, which can simultaneously detect multiple target molecules on the same tissue section sample to achieve different protein co-expression and co-localization analysis. In this experimental protocol, paraffin-embedded tissue sections of lung squamous carcinoma of clinical origin were subjected to multiplex immunohistochemical staining. By optimizing the experimental protocol, multiplex immunohistochemical staining of labeled target cells and proteins was achieved, solving the problem of autofluorescence and channel crosstalk in lung tissues. In addition, multiplex immunohistochemical staining is widely used in the experimental validation of tumor-related, high-throughput sequencing, including single-cell sequencing, proteomics, and tissue space sequencing, providing intuitive and visual pathology validation results.

Author Spotlight: Enhancing Multicolor Fluorescence Localization in Lung Carcinoma Sample

This experimental protocol describes and optimizes a multiplex immunohistochemistry (IHC) staining method, mainly by optimizing single-channel antibody incubation conditions and adjusting the settings of antibodies and channels to solve the problems of autofluorescence and channel crosstalk in lung cancer tissues of clinical origin.

Lung cancer is the leading cause of malignant tumor-related morbidity and mortality all over the world, and the complex tumor microenvironment has been ...

Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic purposes. However, the visualization and quantification of multiple antigens in a single tissue section using conventional chromogenic IHC is challenging. Multiplexed imaging is especially relevant in lymphoma research and diagnostics, where markers have to be interpreted in the context of a complex tumor microenvironment. Here we describe a protocol for multiplexed fluorescent IHC staining to enable the quantitative assessment of multiple targets in specific cell types of interest in lymphoma.The method covers aspects of antibody validation, antibody optimization, the multiplex optimization with markers of lymphoma subtypes, the staining of tissue microarray (TMA) slides, and the scanning of the slides, followed by data analysis, with specific reference to lymphoma. Using this method, scores for both the mean intensity of a marker of interest and the percentage positivity are generated to facilitate further quantitative analysis. Multiplexing minimizes sample utilization and provides spatial information for each marker of interest.

Here we describe a protocol for multiplex fluorescent immunohistochemical staining and imaging for the simultaneous localization of multiple cancer-associated antigens in lymphoma. This protocol can be extended to the colocalization analysis of biomarkers within all tissue sections.

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic ...

Immunology and Infection

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample that can provide information about antigen coexpression and spatial distribution of the markers. However, a lack of suitable antibodies for formalin-fixed tissues may restrict the nature of markers that can be detected. In addition, the staining method is time-consuming. Here we describe a rapid method to perform multispectral fluorescence imaging on frozen tissues. The method includes the fluorophore combinations used, detailed steps for the staining of mouse and human frozen tissues, and the scanning, acquisition, and analysis procedures. For staining analysis, a commercially available semiautomated multispectral fluorescence imaging system is used. Through this method, up to six different markers were stained and detected in a single frozen tissue section. The machine learning analysis software can phenotype cells that can be used for quantitative analysis. The method described here for frozen tissues is useful for the detection of markers that cannot be detected in FFPE tissues or for which antibodies are not available for FFPE tissues.

We describe a rapid staining method to perform multispectral imaging on frozen tissues.

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample ...

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within ...

Biology

Multiplexed Fluorescent Immunohistochemical Staining of Four Endometrial Immune Cell Types in Recurrent Miscarriage

Immunohistochemistry is the most commonly used method for the identification and visualization of tissue antigens in biological research and clinical diagnostics. It can be used to characterize various biological processes or pathologies, such as wound-healing, immune response, tissue rejection, and tissue-biomaterial interactions. However, the visualization and quantification of multiple antigens (especially for immune cells) in a single tissue section using conventional immunohistochemical (IHC) staining remains unsatisfactory. Hence, multiplexed technologies were introduced in recent years to identify multiple biological markers in a single tissue sample or an ensemble of different tissue samples. 
These technologies can be especially useful in differentiating the changes in immune cell-to-cell interactions within the endometrium between fertile women and women with recurrent miscarriages during implantation. This paper describes a detailed protocol for multiplexed fluorescence IHC staining to investigate the density and clustering of four major immune cell types simultaneously in precisely timed endometrial specimens during embryo implantation. The method includes sample preparation, multiplex optimization with markers for immune cell subtypes, and the scanning of the slides, followed by data analysis, with specific reference to detecting endometrial immune cells. 
Using this method, the density and clustering of four major immune cell types in the endometrium can be simultaneously analyzed in a single tissue section. In addition, this paper will discuss the critical factors and troubleshooting to overcome possible fluorophore interference between the fluorescent probes being applied. Importantly, the results from this multiplex staining technique can help provide an in-depth understanding of the immunologic interaction and regulation during embryo implantation.

Despite the advancements in multiplex immunohistochemistry and multispectral imaging, characterizing the density and clustering of major immune cells simultaneously in the endometrium remains a challenge. This paper describes a detailed multiplex staining protocol and imaging for the simultaneous localization of four immune cell types in the endometrium.

Immunohistochemistry is the most commonly used method for the identification and visualization of tissue antigens in biological research and clinical ...

Multiplex Cyclic Fluorescent Immunohistochemistry

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