Name: Author Spotlight: Enhancing Multicolor Fluorescence Localization in Lung Carcinoma Sample
Uploaded: 2023-11-21T13:40:00
Description: Watch this Scientific Journal Video about Multiplex Immunohistochemistry Staining for Paraffin-embedded Lung Cancer Tissue at JoVE.com

The authors would like to acknowledge members of the Clinical Pathology Research Institute West China Hospital, who contributed technical guidance for quality multiplex immunofluorescence and IHC processing. This protocol was supported by the National Natural Science Foundation of China (82200078).

The protocol is approved by the guidelines of the Ethics Committee of West China Hospital, Sichuan University, China. The lung cancer tissue samples were obtained during surgery in the Center of Lung Cancer at West China Hospital, and informed consent was obtained from each patient.
1. Tissue section preparation
<ol>
	<li>FFPE preparation
	<ol>
		<li>Immerse clinical tissues in neutral formalin solution for more than 72 h.</li>
		<li>Complete the process of dehydrating the t...

Deciphering Tumor Microenvironment Using mIHC in Lung Squamous Carcinoma

<ol>
	<li>Zaidi, A. U., Enomoto, H., Milbrandt, J., Roth, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+fluorescent+in+situ+hybridization+and+immunohistochemical+detection+with+tyramide+signal+amplification.">Dual fluorescent in situ hybridization and immunohistochemical detection with tyramide signal amplification.</a> The Journal of Histochemistry and Cytochemistry. 48 (10), 1369-1375 (2000).</li><li>Lovisa, 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=Epithelial-to-mesenchymal+transition+induces+cell+cycle+arrest+and+parenchymal+damage+in+renal+fibrosis.">Epithelial-to-mesenchymal transition induces cell cycle arrest and parenchymal damage in renal fibrosis.</a> Nature Medicine. 21 (9), 998-1009 (2015).</li><li>Nghiem, P. T., 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+with+pembrolizumab+in+advanced+Merkel-cell+carcinoma.">PD-1 blockade with pembrolizumab in advanced Merkel-cell carcinoma.</a> The New England Journal of Medicine. 374 (26), 2542-2552 (2016).</li><li>Zaretsky, J. 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=Mutations+associated+with+acquired+resistance+to+PD-1+blockade+in+melanoma.">Mutations associated with acquired resistance to PD-1 blockade in melanoma.</a> The New England Journal of Medicine. 375 (9), 819-829 (2016).</li><li>Wang, 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=The+heterogeneous+immune+landscape+between+lung+adenocarcinoma+and+squamous+carcinoma+revealed+by+single-cell+RNA+sequencing.">The heterogeneous immune landscape between lung adenocarcinoma and squamous carcinoma revealed by single-cell RNA sequencing.</a> Signal Transduction and Targeted Therapy. 7 (1), 289 (2022).</li><li>Becheva, Z. R., Gabrovska, K. I., Godjevargova, T. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+between+direct+and+indirect+immunofluorescence+method+for+determination+of+somatic+cell+count.">Comparison between direct and indirect immunofluorescence method for determination of somatic cell count.</a> Chemical Papers. 72, 1861-1867 (2018).</li><li>Niedenberger, B. A., Geyer, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+immunostaining+approaches+to+study+early+male+germ+cell+development.">Advanced immunostaining approaches to study early male germ cell development.</a> Stem Cell Research. 27, 162-168 (2018).</li><li>Granier, 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=Tim-3+expression+on+tumor-infiltrating+PD-1+CD8++T+cells+correlates+with+poor+clinical+outcome+in+renal+cell+carcinoma.">Tim-3 expression on tumor-infiltrating PD-1+CD8+ T cells correlates with poor clinical outcome in renal cell carcinoma.</a> Cancer Research. 77 (5), 1075-1082 (2017).</li><li>Badoual, 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-expressing+tumor-infiltrating+T+cells+are+a+favorable+prognostic+biomarker+in+HPV-associated+head+and+neck+cancer.">PD-1-expressing tumor-infiltrating T cells are a favorable prognostic biomarker in HPV-associated head and neck cancer.</a> Cancer Research. 73 (1), 128-138 (2013).</li><li>Meany, D. L., Hackler, L., Zhang, H., Chan, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tyramide+signal+amplification+for+antibody-overlay+lectin+microarray:+a+strategy+to+improve+the+sensitivity+of+targeted+glycan+profiling.">Tyramide signal amplification for antibody-overlay lectin microarray: a strategy to improve the sensitivity of targeted glycan profiling.</a> Journal of Proteome Research. 10 (3), 1425-1431 (2011).</li><li>Surace, 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=Automated+multiplex+immunofluorescence+panel+for+immuno-oncology+studies+on+formalin-fixed+carcinoma+tissue+specimens.">Automated multiplex immunofluorescence panel for immuno-oncology studies on formalin-fixed carcinoma tissue specimens.</a> Journal of Visualized Experiments: JoVE. (143), 58390 (2019).</li><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=Optimization,+design+and+avoiding+pitfalls+in+manual+multiplex+fluorescent+immunohistochemistry.">Optimization, design and avoiding pitfalls in manual multiplex fluorescent immunohistochemistry.</a> Journal of Visualized Experiments: JoVE. (149), 59915 (2019).</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>Mansfield, J. R. <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:+a+review+of+its+technical+aspects+and+applications+in+anatomic+pathology.">Multispectral imaging: a review of its technical aspects and applications in anatomic pathology.</a> Veterinary Pathology. 51 (1), 185-210 (2014).</li><li>Gustavson, M. 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=Development+of+an+unsupervised+pixel-based+clustering+algorithm+for+compartmentalization+of+immunohistochemical+expression+using+Automated+QUantitative+Analysis.">Development of an unsupervised pixel-based clustering algorithm for compartmentalization of immunohistochemical expression using Automated QUantitative Analysis.</a> Applied Immunohistochemistry &amp; Molecular Morphology. 17 (4), 329-337 (2009).</li><li>Lu, 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=Comparison+of+biomarker+modalities+for+predicting+response+to+PD-1/PD-L1+checkpoint+blockade:+a+systematic+review+and+meta-analysis.">Comparison of biomarker modalities for predicting response to PD-1/PD-L1 checkpoint blockade: a systematic review and meta-analysis.</a> JAMA Oncology. 5 (8), 1195-1204 (2019).</li><li>Humphries, M. P., Maxwell, P., Salto-Tellez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=QuPath:+The+global+impact+of+an+open+source+digital+pathology+system.">QuPath: The global impact of an open source digital pathology system.</a> Computational and Structural Biotechnology Journal. 19, 852-859 (2021).</li><li>Bankhead, 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=QuPath:+Open+source+software+for+digital+pathology+image+analysis.">QuPath: Open source software for digital pathology image analysis.</a> Scientific Reports. 7 (1), 16878 (2017).</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>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+4,+Experimental+Pathology+Techniques+of+Respiratory+System.">Chapter 4, Experimental Pathology Techniques of Respiratory System.</a> Atlas of Experimental Pathology Techniques. , 125-126 (2012).</li><li>Wilson, C. 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=Challenges+and+opportunities+in+the+statistical+analysis+of+multiplex+immunofluorescence+data.">Challenges and opportunities in the statistical analysis of multiplex immunofluorescence data.</a> Cancers. 13 (12), 3031 (2021).</li><li>Mori, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+the+tumor+immune+microenvironment+with+Tyramide-based+multiplex+immunofluorescence.">Characterizing the tumor immune microenvironment with Tyramide-based multiplex immunofluorescence.</a> Journal of Mammary Gland Biology and Neoplasia. 25 (4), 417-432 (2020).</li><li>Mansfield, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+context+in+epigenetics:+quantitative+multicolor+imaging+and+automated+per-cell+analysis+of+miRNAs+and+their+putative+targets.">Cellular context in epigenetics: quantitative multicolor imaging and automated per-cell analysis of miRNAs and their putative targets.</a> Methods. 52 (4), 271-280 (2010).</li></ol>

mIHC is an indispensable experimental technique in the field of scientific research for the quantitative and spatial analysis of multiple protein markers at the single-cell level in a single tissue section, providing intuitive and accurate data for the study of disease pathology by focusing on the detailed tissue structure and cellular interactions in the context of the original tissue. The widespread adoption of mIHC technology will require optimized and effective experimental protocols. To address the problems that may...

Tyramine signal amplification (TSA), which has a history of more than 20 years, is a class of assay techniques that use horseradish peroxidase (HRP) for high-density in situ labeling of target antigens and is widely applied in enzyme-linked immunosorbent assays (ELISAs), in situ hybridization (ISH), immunohistochemistry (IHC), and other techniques for the detection of biological antigens, substantially improving the sensitivity of the detected signal1. Opal polychromatic staining based on TSA technology has been recently developed and widely used in several studies2,3,4,5. Traditional immunofluorescence (IF) staining provides researchers with an easy tool for the detection and comparison of the distribution of proteins in the cells and tissues of various model organisms. It is based on antibody-/antigen-specific binding and includes direct and indirect approaches6. Direct immunostaining involves the use of a fluorophore-conjugated primary antibody against the antigen of interest, which enables direct fluorescent detection using a fluorescence microscope. The indirect immunostaining approach involves the application of a fluorophore-conjugated secondary antibody against the unconjugated primary antibody6,7.
Traditional, single-label, immunofluorescence staining methods can stain only one, two, or in some cases, three antigens in tissues, which is a major limitation in mining the rich information contained in tissue sections. The interpretation of quantitative results often depends on visual observation and accurate quantification by imaging software, such as ImageJ. There are technical limitations, such as antibody species restriction, weak fluorescent label signals, and fluorescent dye color overlap (Table 1). The Opal multiplex IHC (mIHC) technique is based on TSA derivation, which allows multiplex staining and differential labeling of more than 7-9 antigens on the same tissue section, with no restriction on the origin of the primary antibody, but requires high specificity of the corresponding antibody against the antigen. The staining procedure is similar to that of normal immunofluorescence staining, except for two differences: each round of staining involves the use of only one antibody and an antibody elution step is added. Antibodies bound to the antigen by noncovalent bonds can be removed by microwave elution, but the TSA fluorescent signal bound to the surface of the antigen by covalent bonds is retained.
Activated tyramine (T) molecules labeled with the dye are highly enriched at the target antigen, allowing efficient amplification of the fluorescent signal. This allows direct labeling of the antigen without antibody interference, and then multicolor labeling can be achieved after multiple staining cycles8,9,10 (Figure 1). Although this technology produces reliable and accurate images for the study of disease, the creation of a useful multiplex fluorescent immunohistochemistry (mfIHC) staining strategy can be time-consuming and exacting due to the need for extensive optimization and design. Therefore, this multiplex panel protocol has been optimized in an automated IHC stainer with a shorter staining time than that of the manual protocol. This approach can be directly applied and adapted by any researcher for immuno-oncology studies on human formalin-fixed and paraffin-embedded (FFPE) tissue samples11. Moreover, the methods for slide preparation, antibody optimization, and multiplex design will be helpful in obtaining robust images that represent accurate cellular interactions in situ and to shorten the optimization period for manual analysis12.
The mfIHC mainly includes image acquisition and data analysis. In terms of image acquisition, multicolor-labeled complex-stained samples need to be detected with professional spectral imaging equipment to identify the various mixed color signals and obtain high signal-to-noise images without interference from tissue autofluorescence. Current equipment for spectral imaging mainly includes spectral confocal microscopes and multispectral tissue imaging systems. The multispectral tissue imaging system is a professional imaging system designed for the quantitative analysis of tissue sections, and its most important feature is the acquisition of image spectral information, which provides both morphological structure and optical mapping information of biological tissue samples13,14. Any pixel in the spectral image contains a complete spectral curve, and each dye (including autofluorescence) has its corresponding characteristic spectrum, which enables the complete recording and accurate identification of mixed and overlapping multilabel signals.
In terms of data analysis, multicolor-labeled samples are extremely complex due to the morphological structure and constituent cells of the tissue samples. Ordinary software cannot automatically identify different tissue types. Hence, intelligent quantitative tissue analysis software is used for quantitative analysis of antigen expression in specific regions15,16,17,18.
Above all, multilabel immunofluorescence staining fused with multispectral imaging and quantitative pathology analysis technology has the advantages of a large number of detection targets, effective staining, and accurate analysis, and it can therefore significantly improve the accuracy of histomorphological analysis, reveal the spatial relationship between proteins with cellular-level resolution, and help to mine richer and more reliable information from tissue section samples19 (Table 1).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD8</td>
			<td>Abcam</td>
			<td>ab237709</td>
			<td>Primary antibody, 1/100, PH9</td>
		</tr>
		<tr>
			<td>Anti-CD68</td>
			<td>Abcam</td>
			<td>ab955</td>
			<td>Primary antibody, 1/300, PH9</td>
		</tr>
		<tr>
			<td>Anti-CK5/6</td>
			<td>Millipore</td>
			<td>MAB1620</td>
			<td>Primary antibody, 1/150, PH9</td>
		</tr>
		<tr>
			<td>Anti-HMGCS1</td>
			<td>GeneTex</td>
			<td>GTX112346</td>
			<td>Primary antibody, 1/300, PH6</td>
		</tr>
		<tr>
			<td>Animal nonimmune serum</td>
			<td>MXB Biotechnologies</td>
			<td>SP KIT-B3</td>
			<td>Antigen blocking</td>
		</tr>
		<tr>
			<td>Fluormount-G</td>
			<td>SouthernBiotech</td>
			<td>0100-01</td>
			<td>Anti-fluorescent burst</td>
		</tr>
		<tr>
			<td>Opal PolarisTM 7-Color Manual IHC Kit</td>
			<td>Akoya</td>
			<td>NEL861001KT</td>
			<td>Opal mIHC Staining</td>
		</tr>
		<tr>
			<td>Wash Buffer</td>
			<td>Dako</td>
			<td>K8000/K8002/K8007/K8023</td>
			<td>Washing the tissues slides</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HALO</td>
			<td></td>
			<td></td>
			<td>intelligent quantitative tissue analysis software, paid software</td>
		</tr>
		<tr>
			<td>inForm</td>
			<td></td>
			<td></td>
			<td>intelligent quantitative tissue analysis software, paid software</td>
		</tr>
		<tr>
			<td>PerkinElmer Vectra</td>
			<td></td>
			<td></td>
			<td>multispectral tissue imaging systems, fully automatic scanning of tissue slides.</td>
		</tr>
		<tr>
			<td>QuPath 0.3.2</td>
			<td></td>
			<td></td>
			<td>intelligent quantitative tissue analysis software, open source software, used in this experiment.</td>
		</tr>
	</tbody>
</table>

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.

Multiplex Immunohistochemistry Staining for Paraffin-embedded Lung Cancer Tissue

We optimized the matching scheme of CD8 primary antibody and fluorophores. Both sets of fluorescence results corresponded to exactly the same antibody incubation conditions in the experimental group, except for the change in the antibody-matched fluorophore. As shown in Figure 2, there was a significant difference in the channel matching of CD8+ T cells with fluorophore 480 and fluorophore 690. The channel with fluorophore 690 shows an extremely strong fluorescent background-the large area o...

Watch this Scientific Journal Video about Multiplex Immunohistochemistry Staining for Paraffin-embedded Lung Cancer Tissue at JoVE.com

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 ...