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 tissues using xylene and graded alcoholic solutions20.</li>
		<li>Perform manual paraffin embedding and tissue sectioning at 4 &#181;m section thickness. Use adhesion microscope slides to ensure that the tissue slices do not fall out.</li>
		<li>Bake the slides in an oven at 65 &#176;C for 2 h to ensure that the tissue and slides are dry. Store in a slide box at room temperature.</li>
	</ol>
	</li>
	<li>Tissue section pretreatment
	<ol>
		<li>Pretreat the tissue slides in an oven at 65 &#176;C for 5 min, and then sequentially immerse in xylene solutions for 2 x 15 min, anhydrous ethanol solutions for 2 x 15 min, 90% ethanol solution for 10 min, 85% ethanol solution for 10 min, 80% ethanol solution for 10 min, and 75% ethanol solution for 10 min, followed by washing the slides with sterilized water for 3 x 1 min. 
		&#8203;NOTE: This experimental technique can only be used for FFPE tissue, not for frozen or fresh tissue, as the multiple high-temperature antigen repair process causes the tissue to fall off the slide.</li>
	</ol>
	</li>
</ol>
2. Optimization of primary antibody
NOTE: Conventional IHC experiments were used to determine the incubation conditions for individual antibodies, mainly including antibody concentration and antigen repair conditions. Refer to the conditions in the antibody instruction manual.
<ol>
	<li>Use an antibody concentration slightly higher than the recommended concentration range and intermediate values.</li>
	<li>Optimize the antigen retrieval buffer routines PH6 and PH9 according to the location of protein expression. Use PH9 for proteins expressed in the nucleus and use either routine (PH6 or PH9) for other proteins.</li>
</ol>
3. mIHC staining method
NOTE: Opal mIHC staining is one of the available mIHC methods. In this experimental 5-color protocol, as each tissue sample needs to be stained with four antibodies, four primary antibody incubations, secondary antibody incubations, and TSA signal amplification chromogenic incubations, as well as five antigen restorations are needed. Finally, 4&#39;6-diamidino-2-phenylindole (DAPI) staining and anti-fluorescent bursting agent sealing are performed.
<ol>
	<li>Main reagent preparation 
	NOTE: Determine the amount of reagent to be added according to the size of the tissue slides. Use enough volume to completely cover the tissue section (generally, 50-150 &#181;L per slide). The working solutions required for the experiment are prepared as follows:
	<ol>
		<li>Wash buffer working solution: dilute 20x Wash buffer at 1:20 with double-distilled water, and store at room temperature.</li>
		<li>PH6 buffer working solution: Dilute 100x PH6 buffer at 1:100 with double-distilled water. Prepare before use and store at room temperature.</li>
		<li>PH9 buffer working solution: Dilute 50x PH9 buffer at 1:50 with double-distilled water. Prepare before use and store at room temperature.</li>
		<li>Polymer HRP: Prepare this ready-to-use solution only for primary antibodies of rabbit and mouse origin.</li>
		<li>Fluorophore working solution: Reconstitute each fluorophore powder (except fluorophore 780) in 75 &#181;L of DMSO. Before each procedure, dilute the fluorophore in 1x amplification diluent at 1:100. Discard any unused portion of the fluorophore working solution.</li>
		<li>Fluorophore 780 Working Solution: Reconstitute TSA-DIG in 75 &#181;L of DMSO and the fluorophore powder 780 in 300 &#181;L of double-distilled water. Before the procedure, dilute TSA-DIG in 1x amplification diluent at 1:100, and dilute fluorophore 780 with Ab diluent at 1:25.</li>
		<li>DAPI working solution: Dilute the DAPI solution at 1:50 with double-distilled water or PBS. Prepare before use and store at 4 &#176;C for no longer than 48 h. 
		NOTE. Wash the slides only with double-distilled water and freshly prepared wash buffer working solution throughout this multilabel fluorescence staining experiment. Tissue sections must not come into contact with tap water; contaminants in the tap water can adhere to the tissue sections and cause autofluorescence. Keep the samples away from light throughout the experiment.</li>
	</ol>
	</li>
	<li>Antigen retrieval
	<ol>
		<li>Place the slides in a staining and repair box and fill it with PH6 or PH9 buffer working solution, cover with the lid, and heat in a microwave oven for 2 x 8 min at 100% power. 
		&#8203;NOTE: Add double-distilled water to the repair box during the heating process to prevent excessive evaporation that can cause the slices to dry out.</li>
		<li>Allow the slides to cool at room temperature before proceeding (30-60 min).</li>
		<li>Wash the slides 3 x 2 min with double-distilled water. Use a hydrophobic barrier pen to encircle the tissue section on the slide.</li>
	</ol>
	</li>
	<li>Blocking
	<ol>
		<li>Immerse tissue sections in wash buffer, cover with blocking solution, and incubate slides in a humidified chamber at room temperature for 10 min.</li>
	</ol>
	</li>
	<li>Primary antibody incubation
	<ol>
		<li>Remove the blocking solution from the slides and cover the tissue sections with the primary antibody working solution. Incubate the slides in a humidified chamber for 1 h at 37 &#176;C in the incubator or overnight at 4 &#176;C in the refrigerator. 
		NOTE: Do not let the slides dry out.</li>
	</ol>
	</li>
	<li>Introduction of Polymer HRP
	<ol>
		<li>Wash the slides in wash buffer working solution for 3 x 2 min. Add Polymer HRP directly dropwise to tissue sections and incubate for 15 min at room temperature. 
		NOTE: For slides stored in the refrigerator, keep them at room temperature for approximately 30 min before washing to remove the primary antibody.</li>
	</ol>
	</li>
	<li>Tyramine signal amplification (TSA) generation
	<ol>
		<li>Wash the slide with wash buffer working solution for 3 x 2 min, add fluorophore working solution directly dropwise to the tissue sections, and incubate for 10 min at room temperature. Wash the slide with wash buffer working solution for 3 x 2 min.</li>
	</ol>
	</li>
	<li>Special procedure for fluorophore 780 
	NOTE: Fluorophore 780 is weak and is routinely placed last in the color-matching scheme of the entire experiment. Fluorophore 780 is not normally used in this experimental protocol, but because of its specificity, its experimental steps are listed.
	<ol>
		<li>Introduction of TSA-DIG
		<ol>
			<li>Wash the slide with wash buffer working solution for 3 x 2 min, add TSA-Drg working solution dropwise to the tissue sections, and incubate for 10 min at room temperature.</li>
		</ol>
		</li>
		<li>Microwave treatment
		<ol>
			<li>Wash the slides with wash buffer working solution for 3 x 2 min.</li>
			<li>Place the slides in a staining and repair box, fill it with repair solution, cover with the lid, and heat in a microwave oven for 2 x 8 min at 100% power. Allow the slides to cool at room temperature before proceeding (30-60 min).</li>
			<li>Wash the slides for 3 x 2 min with double-distilled water.</li>
		</ol>
		</li>
		<li>Fluorophore 780 signal generation
		<ol>
			<li>Immerse the sections in wash buffer, cover with fluorophore 780 working solution, and incubate the slides in a humidified chamber for 1 h at room temperature.</li>
			<li>Wash the slides with wash buffer working solution for 3 x 2 min. 
			NOTE: DO NOT perform microwave treatment after this step.</li>
		</ol>
		</li>
		<li>Use fluorophore 780 as the last step of the entire workflow and then stain nuclei directly with the DAPI working solution. 
		NOTE: Skip step 3.7 if not using fluorophore 780.</li>
	</ol>
	</li>
	<li>Microwave treatment 
	NOTE: This microwave step strips the primary-secondary HRP complex and re-exposes antigens, allowing the introduction of the next primary antibody.
	<ol>
		<li>Place the slides in a staining and repair box, fill it with PH6 or PH9 buffer working solution, cover with the lid, and heat in a microwave oven for 2 x 8 min at 100% power.</li>
		<li>Allow the slides to cool at room temperature before proceeding (30-60 min). Wash the slides 3 x 1 min with double-distilled water.</li>
		<li>Dip slides in wash buffer working solution for 2 min. Perform the next antibody incubation.</li>
	</ol>
	</li>
	<li>Next antibody incubation
	<ol>
		<li>Repeat steps 3.2-3.8 (except step 3.7).</li>
	</ol>
	</li>
	<li>Cell nuclear staining and tissue sealing
	<ol>
		<li>Cover the tissue sections with the DAPI working solution and incubate the slides in a humidified chamber for 5 min at room temperature. Wash the slides for 3 x 2 min with double-distilled water.</li>
		<li>Remove water droplets from the slides, add 10-20 &#181;L of anti-fluorescence quencher to each slide, and seal the slides with a microscope cover glass.</li>
		<li>Store the finished slides at 4 &#176;C, protected from light, for more than 1 month. 
		&#8203;NOTE: Take care to avoid air bubbles.</li>
	</ol>
	</li>
</ol>
4. Set up the negative control
<ol>
	<li>Process the negative control slides like the tissue-containing slides but without the addition of reagents such as primary antibodies, secondary antibodies, TSA reagents, and DAPI, which are used to remove tissue autofluorescence when scanning the film.</li>
</ol>
5. Fully automatic scanning of tissue slides
NOTE: The equipment used for spectral imaging is a fully automated multispectral tissue quantification analyzer, and imaging and analysis visualization of 5-color slides can be performed using the referenced system (see Table of Materials). The system uses multispectral imaging for quantitative unmixing of multiple fluorophores and tissue autofluorescence.
<ol>
	<li>Manually set the exposure time and scanning program for each fluorescence channel and confirm that the instrument has completed the fully automatic exposure and scanning of the tissue slides. 
	&#8203;NOTE: The slide surface must be clean and free of water film. Be careful with the amount of sealer: too much will cause the coverslip to slip and the instrument to report an error.</li>
</ol>
6. Analysis of fluorescence
<ol>
	<li>Double-click the software (see the Table of Materials), drag the multicolor fluorescence image file obtained from the original scan into the software, and set the image type to fluorescence.</li>
	<li>Click the Brightness &#38; contrast button and check the channels on the panel. Click outside and then on the channel to select the fluorescence channel of interest. Drag the Min display and Max display to adjust the brightness and contrast of each channel.</li>
	<li>After analysis, determine the view to be saved, click on Show slide overview and Show cursor location to remove these two contents, keep the scale bar, and click File | Export snapshot | Current viewer content to export a png file. 
	NOTE: Every fluorescence channel and a merged figure must be exported for future analysis.</li>
	<li>For further analysis of target cell positivity, use cell identification and segmentation software21 (see Table of Materials).</li>
</ol>

Deciphering Tumor Microenvironment Using mIHC in Lung Squamous Carcinoma

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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. 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All authors declare that there are no conflicts of interest.

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.

Author Spotlight: Enhancing Multicolor Fluorescence Localization in Lung Carcinoma Sample

Multiplex Immunohistochemistry Staining for Paraffin-embedded Lung Cancer Tissue

Our research focused on optimizing the mIHC protocol on paraffin section obtained from clinic sources. We aimed to effectively address autofluorescence and autofluorescence channel crosstalk issues in lung tissue. The current experimental challenges we are facing involve addressing the issues of autofluorescence and channel crosstalk in lung tissue, whereby achieving successful multicolor fluorescence localization they need for labeled target cells and proteins.Our highly efficient multiple antigen in situ labeling method, which requires less than three hours per antibody, seamlessly integrates a multispectral imaging system and a specialized pathology software. This optimized mIHC protocol is easily adaptable for use in other research laboratories.

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

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 ...

multiplex-immunohistochemistry-staining-for-paraffin-embedded-lung

Research

JoVE Journal

Cancer Research

1.2K Views.  Sichuan University. Our research focused on optimizing the mIHC protocol on paraffin section obtained from clinic sources. We aimed to effectively address autofluorescence and autofluorescence channel crosstalk issues in lung tissue. The current experimental challenges we are facing involve addressing the issues of autofluorescence and channel crosstalk in lung tissue, whereby achieving successful multicolor fluorescence localization they need for labeled target cells and proteins.Our highly efficient mul...