Multiplex Immunohistochemistry Staining for Paraffin-embedded Lung Cancer Tissue

Summary

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.

Abstract

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.

Introduction

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 studies^2,3,4,5. 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 antibody^6,7.

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 cycles^8,9,10 (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 samples^13,14. 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 regions^15,16,17,18.

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).

Protocol

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

1. FFPE preparation
   1. Immerse clinical tissues in neutral formalin solution for more than 72 h.
   2. Complete the process of dehydrating the tissues using xylene and graded alcoholic solutions²⁰.
   3. Perform manual paraffin embedding and tissue sectioning at 4 µm section thickness. Use adhesion microscope slides to ensure that the tissue slices do not fall out.
   4. Bake the slides in an oven at 65 °C for 2 h to ensure that the tissue and slides are dry. Store in a slide box at room temperature.
2. Tissue section pretreatment
   1. Pretreat the tissue slides in an oven at 65 °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.
      ​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.

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.

1. Use an antibody concentration slightly higher than the recommended concentration range and intermediate values.
2. 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.

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, 4'6-diamidino-2-phenylindole (DAPI) staining and anti-fluorescent bursting agent sealing are performed.

1. 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 µL per slide). The working solutions required for the experiment are prepared as follows:
   1. Wash buffer working solution: dilute 20x Wash buffer at 1:20 with double-distilled water, and store at room temperature.
   2. PH6 buffer working solution: Dilute 100x PH6 buffer at 1:100 with double-distilled water. Prepare before use and store at room temperature.
   3. PH9 buffer working solution: Dilute 50x PH9 buffer at 1:50 with double-distilled water. Prepare before use and store at room temperature.
   4. Polymer HRP: Prepare this ready-to-use solution only for primary antibodies of rabbit and mouse origin.
   5. Fluorophore working solution: Reconstitute each fluorophore powder (except fluorophore 780) in 75 µL of DMSO. Before each procedure, dilute the fluorophore in 1x amplification diluent at 1:100. Discard any unused portion of the fluorophore working solution.
   6. Fluorophore 780 Working Solution: Reconstitute TSA-DIG in 75 µL of DMSO and the fluorophore powder 780 in 300 µL 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.
   7. DAPI working solution: Dilute the DAPI solution at 1:50 with double-distilled water or PBS. Prepare before use and store at 4 °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.
2. Antigen retrieval
   1. 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.
      ​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.
   2. Allow the slides to cool at room temperature before proceeding (30-60 min).
   3. Wash the slides 3 x 2 min with double-distilled water. Use a hydrophobic barrier pen to encircle the tissue section on the slide.
3. Blocking
   1. Immerse tissue sections in wash buffer, cover with blocking solution, and incubate slides in a humidified chamber at room temperature for 10 min.
4. Primary antibody incubation
   1. 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 °C in the incubator or overnight at 4 °C in the refrigerator.
      NOTE: Do not let the slides dry out.
5. Introduction of Polymer HRP
   1. 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.
6. Tyramine signal amplification (TSA) generation
   1. 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.
7. 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.
   1. Introduction of TSA-DIG
      1. 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.
   2. Microwave treatment
      1. Wash the slides with wash buffer working solution for 3 x 2 min.
      2. 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).
      3. Wash the slides for 3 x 2 min with double-distilled water.
   3. Fluorophore 780 signal generation
      1. 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.
      2. Wash the slides with wash buffer working solution for 3 x 2 min.
         NOTE: DO NOT perform microwave treatment after this step.
   4. 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.
8. Microwave treatment
   NOTE: This microwave step strips the primary-secondary HRP complex and re-exposes antigens, allowing the introduction of the next primary antibody.
   1. 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.
   2. Allow the slides to cool at room temperature before proceeding (30-60 min). Wash the slides 3 x 1 min with double-distilled water.
   3. Dip slides in wash buffer working solution for 2 min. Perform the next antibody incubation.
9. Next antibody incubation
   1. Repeat steps 3.2-3.8 (except step 3.7).
10. Cell nuclear staining and tissue sealing
    1. 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.
    2. Remove water droplets from the slides, add 10-20 µL of anti-fluorescence quencher to each slide, and seal the slides with a microscope cover glass.
    3. Store the finished slides at 4 °C, protected from light, for more than 1 month.
       ​NOTE: Take care to avoid air bubbles.

4. Set up the negative control

1. 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.

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.

1. 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.
   ​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.

6. Analysis of fluorescence

1. 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.
2. Click the Brightness & 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.
3. 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.
4. For further analysis of target cell positivity, use cell identification and segmentation software²¹ (see Table of Materials).

Representative Results

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 of red irregular fluorescence within the yellow circle shows color, which causes great interference in the localization analysis of CD8+ T cells (Figure 2C,D). However, the channel with fluorophore 480 shows a very specific CD8 cell membrane positivity and no fluorescent background. Thus, the yellow circles show a very clear positive cell membrane (Figure 2A), which fully illustrates the importance of antibody and fluorescence channel matching in this protocol.

The distribution of macrophages and cytotoxic T cells was examined in non-small cell lung squamous carcinoma tissues, and the expression of the characteristic protein HMGCS1 in tumor cells and immune cells was verified. As shown in Figure 3, the images were derived from QuPath 0.3.2. The mIHC panel was fluorophores 480, 520, 620, 690, and DAPI, and the corresponding antibody markers were CK5/6, a marker for lung squamous carcinoma cells; CD8, a marker for T cells; CD68, a marker for macrophages; and HMGCS1, which is specific for plasma-positive tumor cells. Macrophages and CD8+ T cells showed heavy infiltration and were distributed around and within the squamous carcinoma foci, while HMGCS1 was widely expressed in the squamous carcinoma cell plasma, showing strong tumor cell positivity.

Figure 1: The workflow of multiplex immunohistochemical staining for FFPE tissue. Abbreviation: FFPE = formalin-fixed and paraffin-embedded. Please click here to view a larger version of this figure.

Figure 2: Optimization of the matching protocols for CD8 and fluorophores. There was a significant difference in the channel matching of CD8+ T cells with fluorophore 480 and fluorophore 690. Scale bars = 50 µm. Please click here to view a larger version of this figure.

Figure 3: Multiplexed immunohistochemical staining in lung squamous carcinoma. The distribution of macrophages and cytotoxic T cells in non-small cell lung squamous carcinoma tissues and the expression of the characteristic protein HMGCS1 in tumor cells and immune cells. CK5/6 is a marker for lung squamous carcinoma cells; CD8 is a marker for T cells; CD68 is a marker for macrophages; and HMGCS1 is specific to tumor cell plasma-positive cells. Scale bars = 50 µm. Please click here to view a larger version of this figure.

	Multiplex Immunohistochemistry technique			Traditional immunofluorescence technique
Number of Molecular Targets	Unlimited (up to 9 kinds of dyeing at once, including DAPI)			General marking 3-4 kinds (including DAPI)
Image Acquisition	High staining resolution and staining specificity, signaling amplification, strong signal intensity, longer quenching time, no background effect, and much higher signal-to-noise ratio at each target site.			Weak brightness, easy quenching, mutual influence between various antibodies, high background
Data Analysis	Area specificity, cell segmentation, single-cell spatial information			Naked eye observation and accurate quantification by ImageJ

Table 1: Comparison between Opal multi-labeling immunofluorescence technology and traditional immunofluorescence technology.

Discussion

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 arise in the experiments, we present the following considerations and solutions.

First, according to the principle of matching antigen and fluorescein-labeled antibody in multi-color experiments, weakly expressed antigen is matched with strongly fluorescein-labeled antibody, and strongly expressed antigen is matched with weakly fluorescein-labeled antibody. Combined with the results of IHC experiments of single antibody-labeled antigens, the intensity of expression of the target protein is determined, and the matching of antibody and fluorescein is adjusted, finally determining the matching scheme of antibody and fluorescein required in multi-color labeling experiments. Adjacent fluorescence channels with different protein expression levels of the antibody can solve the problem of adjacent fluorescence channel string color. Second, the software settings for detecting fluorescence signals were set using the Akoya Biosciences imaging system for imaging stained slides, and the fluorescence acquisition parameters were set by using the multispectral scanning software Phenochart 1.0, which allows checking the fluorescence signal balance²². It is recommended that the exposure time be within 100 ms for a single fluorescence channel and should be more homogeneous and not higher than 200 ms for multiple fluorescence channels. The fluorescence parameters are adjusted based on the IHC-optimized antibody incubation conditions.

Third, the problem of autofluorescence can be analyzed by setting a blank control, combined with the characteristic spectral detection of multispectral imaging. This can identify the mixed color signals and, for formalin-fixed paraffin-embedded samples, improve the signal-to-noise ratio of the images by approximately an order of magnitude by removing tissue autofluorescence²³. Fourth, to address tissue-nonspecific chromogenicity, antibodies with poor tissue specificity should be used with animal nonimmune serum as a blocking solution, usually added before the primary antibody. The selected serum species is generally the same as the source of the secondary antibody species, which can reduce nonspecific chromogenicity. Fifth, it is recommended to set up negative control and positive controls as experimental quality controls. The positive control has the role of checking the quality of reagents, and the negative control has the dual function of verifying the quality of the experimental procedures and removing autofluorescence when scanning slides. The combination can be used to ensure the accuracy of each experiment. Sixth, the slide washing step is very important throughout the experiment. The reagents prepared in the protocol should be used, and the wash time should be used to wash the reagents off the slides at each step to prevent interference with the labeling in the next step.

When the specificity of a single fluorescence channel is poor, resulting in nonspecific positivity and fluorescence crosstalk, users must check the IHC result of the appropriate antibody, confirm the positive result, and then, consider changing the relevant fluorophore. If a single positive fluorescence result is too weak or too strong, it is important to reset the film scanning parameters to the recommended fluorophore exposure time of 10-150 ms.

This multiple labeling method is currently limited to paraffin-embedded tissue samples, as the protein labeling process requires multiple antigenic thermal repairs, which require the tissue to be fixed to prevent breakage. Frozen sections of fresh tissue will not withstand the damage caused by thermal repair, and large amounts of tissue will be lost.

In summary, this multiple antigen in situ labeling method is less time-consuming, with a single antibody labeling process time of less than 3 h. It combines the advantages of a multispectral imaging system and professional pathology analysis software to optimize the mIHC experimental protocol in terms of individual protein labeling conditions and experimental group and fluorescence channel settings.

Materials

Name	Company	Catalog Number	Comments
Reagents
Anti-CD8	Abcam	ab237709	Primary antibody, 1/100, PH9
Anti-CD68	Abcam	ab955	Primary antibody, 1/300, PH9
Anti-CK5/6	Millipore	MAB1620	Primary antibody, 1/150, PH9
Anti-HMGCS1	GeneTex	GTX112346	Primary antibody, 1/300, PH6
Animal nonimmune serum	MXB Biotechnologies	SP KIT-B3	Antigen blocking
Fluormount-G	SouthernBiotech	0100-01	Anti-fluorescent burst
Opal PolarisTM 7-Color Manual IHC Kit	Akoya	NEL861001KT	Opal mIHC Staining
Wash Buffer	Dako	K8000/K8002/K8007/K8023	Washing the tissues slides
Software
HALO			intelligent quantitative tissue analysis software, paid software
inForm			intelligent quantitative tissue analysis software, paid software
PerkinElmer Vectra			multispectral tissue imaging systems, fully automatic scanning of tissue slides.
QuPath 0.3.2			intelligent quantitative tissue analysis software, open source software, used in this experiment.