Name: Video: Author Spotlight: Enhancing Multicolor Fluorescence Localization in Lung Carcinoma Sample
Uploaded: 2023-11-21T13:40:00.000+00:00
Duration: 5 min
Description: 1.5K Views. Sichuan University. 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.

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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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.

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.

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 ...

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

Research

JoVE Journal

Cancer Research

1.5K Views.  Sichuan University. 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.

Video: Author Spotlight: Enhancing Multicolor Fluorescence Localization in Lung Carcinoma Sample

Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinoma Samples

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their relative distribution and their localization in the tissue. This technique has been extensively applied to study the immune infiltrate in various tumor types. The tumor microenvironment is infiltrated by immune cells that are attracted to the tumor site. Different immune cell populations have been found to play different roles in the tumor microenvironment and to have a different impact on the outcome of disease. This manuscript describes the use of multiparameter fluorescence IHC on oropharyngeal squamous cell carcinoma (OPSCC) as an example. This technique can be extended to other tissue samples and cell types of interest. In the presented study, we analyzed the intraepithelial and stromal compartment of a large OPSCC cohort (n = 162). We focused on total T lymphocytes (CD3+), immunosuppressive regulatory T cells (Tregs; i.e., FoxP3+), and T helper 17 (Th17) cells (i.e., IL-17+CD3+) using a nuclear counterstain to distinguish tumor epithelium from stroma. A high number of T cells was found to be correlated with improved disease-free survival in patients with a low number of intratumoral IL-17+ non-T cells. This suggests that IL-17+ non-T cells may be correlated with a poor immune response in OPSCC, which is in agreement with the correlation described between IL-17 and poor survival in cancer patients. Currently, novel multiparameter fluorescence IHC techniques are being developed using up to 7 different fluorochromes and will enable the more precise characterization and localization of immune cells in the tumor microenvironment.

Multiparameter fluorescence immunohistochemistry can be used to assess the number, relative distribution, and localization of immune cell populations in the tumor microenvironment. This manuscript describes the use of this technique to analyze T-cell subpopulations in oropharyngeal cancer.

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their ...

Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung colonization in osteosarcoma (OS) by fluorescence microscopy. This article provides a detailed description of the protocol, and discusses examples of obtaining image data on metastatic growth using widefield or confocal fluorescence microscopy platforms. The flexibility of the PuMA model permits researchers to study not only the growth of OS cells in the lung microenvironment, but also to assess the effects of anti-metastatic therapeutics over time. Confocal microscopy allows for unprecedented, high-resolution imaging of OS cell interactions with the lung parenchyma. Moreover, when the PuMA model is combined with fluorescent dyes or fluorescent protein genetic reporters, researchers can study the lung microenvironment, cellular and subcellular structures, gene function, and promoter activity in metastatic OS cells. The PuMA model provides a new tool for osteosarcoma researchers to discover new metastasis biology and assess the activity of novel anti-metastatic, targeted therapies.

The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope.

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung ...

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

Determination of Protein Expression Level in Cultured Cells by Immunocytochemistry on Paraffin-embedded Cell Blocks

Immunofluorescent staining is currently the method of choice for determination of protein expression levels in cell-culture systems when morphological information is also necessary. The protocol of immunocytochemical staining on paraffin-embedded cell blocks, presented herein, is an excellent alternative to immunofluorescent staining on non-paraffin-embedded fixed cells. In this protocol, a paraffin cell block from HeLa cells was prepared using the thromboplastin-plasma method, and immunocytochemistry was performed for the evaluation of two proliferation markers, CKAP2 and Ki-67. The nuclei and cytoplasmic morphology of the HeLa cells were well preserved in the cell-block slides. At the same time, the CKAP2 and Ki-67 staining patterns in the immunocytochemistry were quite similar to those in immunohistochemical staining in paraffin cancer tissues. With modified cell-culture conditions, including pre-incubation of HeLa cells under serum-free conditions, the effect could be evaluated while preserving architectural information. In conclusion, immunocytochemistry on paraffin-embedded cell blocks is an excellent alternative to immunofluorescent staining.

Currently, immunofluorescent staining on fixed cells is the method of choice for determination of protein expression levels when morphological information is also necessary. This protocol presented herein provides for an alternative method of immunocytochemistry on paraffin-embedded cell blocks.

Immunofluorescent staining is currently the method of choice for determination of protein expression levels in cell-culture systems when morphological ...

Elastic Staining on Paraffin-embedded Slides of pT3N0M0 Gastric Cancer Tissue

The elastic lamina, which is usually located in the sub-mesothelial layer, adjacent to the peritoneum mesothelial cells, is amphiphilic on hematoxylin and eosin (H&amp;E) staining but can be visualized through elastic staining. One of the important benefits of elastic staining is that it makes it easy to determine whether tumor cells have invaded beyond the elastic lamina. This helps in examining the extent of peritoneal surface invasion, thereby distinguishing the pT3 and pT4 stages in gastrointestinal cancer. In this study, we present a protocol to identify elastic fibers in the formalin-fixed, paraffin-embedded pT3N0M0 gastric cancer tissue sections. We prepare 5-&#181;m paraffin sections fixed on slides, and then all the slides are deparaffinated and rehydrated during the staining procedure. Subsequently, all the sections are oxidized by potassium permanganate, bleached by oxalic acid, stained by elastic staining, and counterstained by Van Gieson's. Finally, we examine the effect of staining; elastic lamina is stained blue-black and collagen fibers are stained red, while the cancer cells are stained in varying shades of yellow. We also describe in detail the methods used for determining the positional relationship between cancer cells and elastic lamina. This method is simple, low-cost, and widely applicable in the identification of peritoneal surface invasion in gastrointestinal cancer because of its outstanding selectivity for elastic fibers and authentic results.

Here, we present a protocol of elastic staining to identify elastic fibers in pT3N0M0 gastric cancer tissues on formalin-fixed, paraffin-embedded sections. Subsequently, we describe a method to determine whether tumor cells invade beyond the elastic lamina.

The elastic lamina, which is usually located in the sub-mesothelial layer, adjacent to the peritoneum mesothelial cells, is amphiphilic on hematoxylin and ...

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

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

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

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

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 &#181;m pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as biomarkers with applications in diagnosis, prognosis, classification, state of tumor progression, and cell differentiation state. These analyses of biomarkers are also useful to characterize tumor neurospheres (NS) generated from glioma patients and glioma models. Tumor NS provide a valuable in vitro model to assess different features of the tumor from which they are derived and can more accurately mirror glioma biology. Here we describe a detailed method to analyze biomarkers in tumor NS using immunohistochemistry (IHC) on paraffin-embedded tumor NS.

Neurospheres grown as 3D cultures constitute a powerful tool to study glioma biology. Here we present a protocol to perform immunohistochemistry while maintaining the 3D structure of glioma neurospheres through paraffin embedding. This method enables the characterization of glioma neurosphere properties such as stemness and neural differentiation.

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as ...

Optimization, Design and Avoiding Pitfalls in Manual Multiplex Fluorescent Immunohistochemistry

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

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

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

Radiosensitivity of Cancer Stem Cells in Lung Cancer Cell Lines

The presence of cancer stem cells (CSCs) has been associated with relapse or poor outcomes after radiotherapy. Studying radioresistant CSCs may provide clues to overcoming radioresistance. Voltage-gated calcium channel &#945;2&#948;1 subunit isoform 5 has been reported as a marker for radioresistant CSCs in non-small cell lung cancer (NSCLC) cell lines. Using calcium channel &#945;2&#948;1 subunit as an example of a CSC marker, methods to study the radiosensitivity of CSCs in NSCLC cell lines are presented. CSCs are sorted with putative markers by flow cytometry, and the self-renewal capacity of sorted cells is evaluated by sphere formation assay. Colony formation assay, which determines how many cells lose the ability to generate descendants forming the colony after a certain dose of radiation, is then performed to assess the radiosensitivity of sorted cells. This manuscript provides initial steps for studying the radiosensitivity of CSCs, which establishes the basis for further understanding of the underlying mechanisms.

The presence of cancer stem cells have been associated with relapse or poor outcomes after radiotherapy. This manuscript describes the methods to study the radiosensitivity of cancer stem cells in lung cancer cell lines.

The presence of cancer stem cells (CSCs) has been associated with relapse or poor outcomes after radiotherapy. Studying radioresistant CSCs may provide ...