Name: optimization, design and avoiding pitfalls in manual multiplex fluorescent immunohistochemistry
Uploaded: 2019-07-26T14:30:00
Description: Watch this Scientific Journal Video about Optimization, Design and Avoiding Pitfalls in Manual Multiplex Fluorescent Immunohistochemistry at JoVE.com

The authors would like to thank Ed Stack, previously from Perkin Elmer, for his assistance with setup and optimization of original multiplex staining. The authors would also like to thank Kristen Schmidt from Akoya Biosciences for tips using the analysis software. Research reported in this publication was supported by the National Cancer Institutes of Health under Award Number P30CA046592, K08CA201581(tlf), K08CA234222 (js), R01CA15158807 (mpm), RSG1417301CSM (mpm), R01CA19807403 (mpm), U01CA22414501 (mpm, hc), CA170568 (hc). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Additional support was provided by Jeffery A. Colby Colon Cancer and Tom Liu Memorial&nbsp;Research Funds.

All work has been approved by the University of Michigan&rsquo;s internal review board.
1. Optimizing primary antibodies and slide preparation
<ol>
<li>Determine ideal concentration of antibodies for multiplex using conventional IHC.
<ol>
<li>Test the antibodies for the multiplex by manual conventional immunohistochemistry (IHC)13.</li>
<li>Use specific tissues that have an abundant cell type for each antibody tested such as using tonsil tissue for CD3 antibody testin...

<ol>
	<li>Lazarus, 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=Spatial+and+phenotypic+immune+profiling+of+metastatic+colon+cancer.">Spatial and phenotypic immune profiling of metastatic colon cancer.</a> JCI Insight. 3 (22), (2018).</li><li>Barua, 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+Functional+Spatial+Analysis+Platform+for+Discovery+of+Immunological+Interactions+Predictive+of+Low-Grade+to+High-Grade+Transition+of+Pancreatic+Intraductal+Papillary+Mucinous+Neoplasms.">A Functional Spatial Analysis Platform for Discovery of Immunological Interactions Predictive of Low-Grade to High-Grade Transition of Pancreatic Intraductal Papillary Mucinous Neoplasms.</a> Cancer Informatics. 17, 1176935118782880 (2018).</li><li>Yang, L., Liu, Z., Tan, J., Dong, H., Zhang, X. <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+reveals+hyper+active+TGF-beta+signaling+in+colorectal+cancer.">Multispectral imaging reveals hyper active TGF-beta signaling in colorectal cancer.</a> Cancer Biology &amp; Therapy. 19 (2), 105-112 (2018).</li><li>Carstens, J. 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=Spatial+computation+of+intratumoral+T+cells+correlates+with+survival+of+patients+with+pancreatic+cancer.">Spatial computation of intratumoral T cells correlates with survival of patients with pancreatic cancer.</a> Nature Communications. 8, 15095 (2017).</li><li>Steele, K. E., Brown, C. <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+for+Image+Analysis+of+Tertiary+Lymphoid+Structures+in+Cancer.">Multiplex Immunohistochemistry for Image Analysis of Tertiary Lymphoid Structures in Cancer.</a> Methods In Molecular Biology. 1845, 87-98 (2018).</li><li>Gorris, M. A. 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=Eight-Color+Multiplex+Immunohistochemistry+for+Simultaneous+Detection+of+Multiple+Immune+Checkpoint+Molecules+within+the+Tumor+Microenvironment.">Eight-Color Multiplex Immunohistochemistry for Simultaneous Detection of Multiple Immune Checkpoint Molecules within the Tumor Microenvironment.</a> The Journal Of Immunology. 200 (1), 347-354 (2018).</li><li>Parra, E. R., 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=Validation+of+multiplex+immunofluorescence+panels+using+multispectral+microscopy+for+immune-profiling+of+formalin-fixed+and+paraffin-embedded+human+tumor+tissues.">Validation of multiplex immunofluorescence panels using multispectral microscopy for immune-profiling of formalin-fixed and paraffin-embedded human tumor tissues.</a> Scientific Reports. 7, (2017).</li><li>Katikireddy, K. R., O'Sullivan, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+and+immunofluorescence+procedures+for+protein+analysis.">Immunohistochemical and immunofluorescence procedures for protein analysis.</a> Methods In Molecular Biology. 784, 155-167 (2011).</li><li>Adan, A., Alizada, G., Kiraz, Y., Baran, Y., Nalbant, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry:+basic+principles+and+applications.">Flow cytometry: basic principles and applications.</a> Critical Reviews In Biotechnology. 37 (2), 163-176 (2017).</li><li>Zaritskaya, L., Shurin, M. R., Sayers, T. J., Malyguine, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+flow+cytometric+assays+for+monitoring+cell-mediated+cytotoxicity.">New flow cytometric assays for monitoring cell-mediated cytotoxicity.</a> Expert Review of Vaccines. 9 (6), 601-616 (2010).</li><li>Stack, E. C., Wang, C., Roman, K. A., Hoyt, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+immunohistochemistry,+imaging,+and+quantitation:+a+review,+with+an+assessment+of+Tyramide+signal+amplification,+multispectral+imaging+and+multiplex+analysis.">Multiplexed immunohistochemistry, imaging, and quantitation: a review, with an assessment of Tyramide signal amplification, multispectral imaging and multiplex analysis.</a> Methods. 70 (1), 46-58 (2014).</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=Fully+automated+5-plex+fluorescent+immunohistochemistry+with+tyramide+signal+amplification+and+same+species+antibodies.">Fully automated 5-plex fluorescent immunohistochemistry with tyramide signal amplification and same species antibodies.</a> Laboratory Investigation. 97 (7), 873-885 (2017).</li><li>Phenoptics Research Solutions. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Perkin Elmer Manual. , 32 (2016).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Opal Multiplex IHC Assay Development Guide. , (2014).</li><li>Carey, C. D., 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=Topological+analysis+reveals+a+PD-L1-associated+microenvironmental+niche+for+Reed-Sternberg+cells+in+Hodgkin+lymphoma.">Topological analysis reveals a PD-L1-associated microenvironmental niche for Reed-Sternberg cells in Hodgkin lymphoma.</a> Blood. 130 (22), 2420-2430 (2017).</li></ol>

Intact tissue specimens from solid tumor biopsy and surgical resection remain important diagnostic and predictive tools for disease analysis as well as patient prognosis. Multiplex fluorescent immunohistochemistry (mfIHC) is a novel technique that combines the benefits of immunohistochemistry (IHC), immunofluorescence (IF) and flow cytometry. Previous methods to probe cells in situ have allowed for evaluation of cell-to-cell arrangements in a tissue environment8, however, the low number of epitope...

Visualization of an intact tumor microenvironment (TME) is essential in evaluating not only cellular infiltration in solid malignancies but cell to cell interactions as well. Multiplex fluorescent immunohistochemistry (mfIHC) has emerged as an effective tool for multi-antigen phenotyping of cells in the study of cancer and associated diseases1,2,3,4,5,6,7. This, in combination with novel software and programs designed to analyze large data sets, enables observation of complex interactions between cells1,2,4. The rate limiting factor in data acquisition is often the quality of the stained tissue prior to analysis.
Previous techniques used to phenotype cells in the TME include immunohistochemistry (IHC), immunofluorescence (IF) and flow cytometry all of which present significant limitations. IHC uses formalin fixed paraffin embedded (FFPE) tissue sections which are deparaffinized and rehydrated before being stained by most often one antibody. Use of a horseradish peroxidase (HRP) bound secondary antibody and a chemical reaction allows visualization of a single antigenic epitope8. While IHC is reliable and performed on FFPE tissue which is easy to work with, limitations to the visible light spectrum means only one or two markers can be reliable distinguished and colocalization of antigens quite difficult8. A way to expand available markers and therefore antigens that can be probed on a single section is to change to fluorescence which allows for use of a broader range of the visual spectrum. For IF, frozen or FFPE tissue is transferred onto slides and antibodies used that are conjugated to various fluorophores. While this increases the number of antigens that can be probed, there are several important limitations. First, because each antibody typically only has one fluorophore attached, the brightness is often not strong enough to overcome tissue autofluorescence. It is for this reason, most IF is performed on frozen tissue which is expensive to store and difficult to work with. A limited number of fluorescent tags are available for use due to spectral overlap and cross species reactivity particularly when non-conjugated antibodies are used. Flow cytometry, which consists of fresh tissue processing into a single cell suspension and labeling with fluorescent antibodies has been the gold standard for immunophenotyping for decades9,10. A benefit of flow cytometry is the ability to label multiple antibodies without concern for species cross reactivity as most are conjugated. Because the cells are &ldquo;visualized&rdquo; by a machine and not human eyes, there are far more fluorophores available but this comes with a cost. Compensation must be done manually which can significantly alter results producing false positive and negative populations. The most significant limitation of flow cytometry is that tissue architecture and subsequently all spatial information is lost by the necessity for single cells suspension.
Multiplex fluorescent immunohistochemistry (mfIHC) using an automated fluorescent microscope in combination with novel software combines the benefits of IHC, IF and flow cytometry by allowing multi-antigen tissue staining with signal amplification and retention of spatial relationships without the need for compensation. FFPE tissue is placed on charged slides which, after antigen retrieval, undergo a round of primary antibody application to the target antigen of interest followed by a secondary antibody with an HRP chemical tag, similar to IHC. After placement of the secondary antibody, an HRP specific reaction results in a fluorophore covalently binding to the epitope of interest11. Once the tissue is labeled, another round of heating the slides is completed removing the previously applied primary and secondary antibody complex leaving only the fluorescent tag bound to the tissue epitope11. This allows for multiple antibodies of any species to be reapplied without concern for cross-reactivity11,12. To minimize any need for manual compensation of multiple fluorescent dyes, a collection of fluorophores with little spectral overlap including a nuclear counter stain is used to complete the mfIHC. To account for the autofluorescence encountered with FFPE tissue, software subtracts the autofluorescence from the final image using an image from a blank slide which is possible because of the strength of antigen specific fluorescence following fluorophore signal amplification. Using novel programs designed for large data sets, cell locations can be identified and analyzed for spatial context1,2,4. The most significant limitation of this technique is optimization time. A detailed methodology with instructions for experimental design and staining and imaging strategy is found here. mfIHC will be useful for laboratories that do not currently have an optimized automated staining system that would like to better understand the spatial context of cell-to-cell interactions in intact FFPE tissue using the manual technique.

<table>
	<tbody>
		<tr>
			<td>100% ethanol</td>
			<td>Fisher</td>
			<td>HC8001GAL</td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>Fisher</td>
			<td>HC10001GL</td>
			<td></td>
		</tr>
		<tr>
			<td>95% ethanol</td>
			<td>Fisher</td>
			<td>HC11001GL</td>
			<td></td>
		</tr>
		<tr>
			<td>Analysis software</td>
			<td>Akoya Biosciences</td>
			<td>CLS135783</td>
			<td>inForm version 2.3.0</td>
		</tr>
		<tr>
			<td>Antifade mountant</td>
			<td>ThermoFisher</td>
			<td>P36961</td>
			<td>ProLong Diamond</td>
		</tr>
		<tr>
			<td>Blocking solution</td>
			<td>Vector</td>
			<td>SP-6000</td>
			<td>Bloxall</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma Life Sciences</td>
			<td>A9647-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover Slips</td>
			<td>Fisher</td>
			<td>12-548-5E</td>
			<td></td>
		</tr>
		<tr>
			<td>Delicate task wipe</td>
			<td>Kimberly-Clark</td>
			<td>34120</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent diluent</td>
			<td>Akoya Biosciences</td>
			<td>ARD1A01EA</td>
			<td>Opal TSA diluent</td>
		</tr>
		<tr>
			<td>Fluorophore 520</td>
			<td>Akoya Biosciences</td>
			<td>FP1487001KT</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>Fluorophore 540</td>
			<td>Akoya Biosciences</td>
			<td>FP1494001KT</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>Fluorophore 570</td>
			<td>Akoya Biosciences</td>
			<td>FP1488001KT</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>Fluorophore 620</td>
			<td>Akoya Biosciences</td>
			<td>FP1495001KT</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>Fluorophore 650</td>
			<td>Akoya Biosciences</td>
			<td>FP1496001KT</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>Fluorophore 690</td>
			<td>Akoya Biosciences</td>
			<td>FP1497001KT</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>Fluorophore DAPI</td>
			<td>Akoya Biosciences</td>
			<td>FP1490</td>
			<td>3 drops in TBST or PBS</td>
		</tr>
		<tr>
			<td>Heat resistant box</td>
			<td>Tissue-Tek</td>
			<td>25608-904</td>
			<td>Plastic slide box-green</td>
		</tr>
		<tr>
			<td>Humidified Chamber</td>
			<td>Ibi Scientific</td>
			<td>AT-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization oven</td>
			<td>FisherBiotech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrophobic barrier pen</td>
			<td>Vector</td>
			<td>H-4000</td>
			<td>ImmEdge</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Perkin Elmer</td>
			<td>CLS140089</td>
			<td>Mantra quantitative pathology workstation</td>
		</tr>
		<tr>
			<td>Microwave</td>
			<td>Panasonic</td>
			<td>NN-A661S</td>
			<td>with inverter technology</td>
		</tr>
		<tr>
			<td>Neutral buffered formalin</td>
			<td>Fisher Scientific</td>
			<td>SF100-4</td>
			<td>10% neutral buffered formalin</td>
		</tr>
		<tr>
			<td>pH 6 antigen retrieval buffer</td>
			<td>Akoya Biosciences</td>
			<td>AR600</td>
			<td>AR6</td>
		</tr>
		<tr>
			<td>pH 9 antigen retrieval buffer</td>
			<td>Akoya Biosciences</td>
			<td>AR900</td>
			<td>AR9</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Fisher</td>
			<td>BP3994</td>
			<td>PBS</td>
		</tr>
		<tr>
			<td>Plastic slide box</td>
			<td>Tissue-Tek</td>
			<td>25608-906</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic wrap</td>
			<td>Fisher</td>
			<td>NC9070936</td>
			<td></td>
		</tr>
		<tr>
			<td>Polysorbate 20</td>
			<td>Fisher</td>
			<td>BP337-800</td>
			<td>Tween 20</td>
		</tr>
		<tr>
			<td>Primary antibody CD163</td>
			<td>Lecia</td>
			<td>NCL-LCD163</td>
			<td>1:400</td>
		</tr>
		<tr>
			<td>Primary antibody CD3</td>
			<td>Dako</td>
			<td>A0452</td>
			<td>1:400</td>
		</tr>
		<tr>
			<td>Primary antibody CD8</td>
			<td>Spring Bio</td>
			<td>M5390</td>
			<td>1:400</td>
		</tr>
		<tr>
			<td>Primary antibody FOXP3</td>
			<td>Dako</td>
			<td>M3515</td>
			<td>1:400</td>
		</tr>
		<tr>
			<td>Primary antibody pancytokeratin</td>
			<td>Cell Signaling</td>
			<td>12653</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Primary antibody PD-L1</td>
			<td>Cell Signaling</td>
			<td>13684</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>Secondary antibody</td>
			<td>Akoya Biosciences</td>
			<td>ARH1001EA</td>
			<td>Opal polymer</td>
		</tr>
		<tr>
			<td>Slide stain set</td>
			<td>Electron Microscopy Sciences</td>
			<td>6254001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris buffered saline</td>
			<td>Corning</td>
			<td>46-012-CM</td>
			<td>TBS</td>
		</tr>
		<tr>
			<td>Vertical slide rack</td>
			<td>Electron Microscopy Sciences</td>
			<td>50-294-72</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Fisher</td>
			<td>X3P1GAL</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The overall process of obtaining a 7-color multiplex assay follows a repetitive pattern. Figure 1 describes the process in a diagrammatic form. Once slides are cut and dried or are received from the laboratory and baked in a hybridization oven at 60 &#176;C for 1 h, then proceed to deparaffinization and rehydration, fix the slides in formalin again followed by antigen retrieval. Each round of multiplexing starts at antigen retrieval and finishes at antibody removal (Figu...

Watch this Scientific Journal Video about Optimization, Design and Avoiding Pitfalls in Manual Multiplex Fluorescent Immunohistochemistry at JoVE.com

Optimization, Design and Avoiding Pitfalls in Manual Multiplex Fluorescent Immunohistochemistry

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 ...

Research

JoVE Journal

Cancer Research

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for ...

Manual Construction of a Tissue Microarray using the Tape Method and a Handheld Microarrayer

The tissue microarray (TMA) is an important research tool in which many formalin fixed paraffin embedded (FFPE) samples can be represented in a single ...

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...

ATAC-Seq Optimization for Cancer Epigenetics Research

The assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) probes deoxyribonucleic acid (DNA) accessibility using the ...

Quantifying Telomere Length Using Non-Radioactive Chemiluminescence Detection

Optimization of Performance Parameters of the TAGGG Telomere Length Assay

Optimization of Performance Parameters of the TAGGG Telomere Length Assay (Video) | JoVE 

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening ...

https://cloudfront.jove.com/CDNSource/teasers/65288.jpg

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

Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment (Video) | JoVE 

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

Deciphering Tumor Microenvironment Using mIHC in Lung Squamous Carcinoma

Multiplex Immunohistochemistry Staining for Paraffin-embedded Lung Cancer Tissue

Author Spotlight: Enhancing Multicolor Fluorescence Localization in Lung Carcinoma Sample

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