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

All work has been approved by the University of Michigan&rsquo;s internal review board.
1. Optimizing primary antibodies and slide preparation
<ol>
<li>Determine ideal concentration of antibodies for multiplex using conventional IHC.
<ol>
<li>Test the antibodies for the multiplex by manual conventional immunohistochemistry (IHC)13.</li>
<li>Use specific tissues that have an abundant cell type for each antibody tested such as using tonsil tissue for CD3 antibody testing.</li>
<li>Use the reference concentration for IHC recommended by the company and then complete IHC with antibody concentrations at the recommended concentrations as well as concentrations above and below the recommended concentration.</li>
</ol>
</li>
<li>Prepare formalin fixed paraffin embedded tissue onto slides.
<ol>
<li>Using a microtome, cut blocks of FFPE tissue at a thickness between 4 and 6 &micro;m and apply to charged slides. NOTE: Using distilled water ensures proper tissue mounting and adherence throughout the multiplex.</li>
<li>Place the slides flat, tissue side up, and allow to dry at 37 &deg;C overnight.</li>
<li>Store slides in a slide box away from extreme temperature until ready to complete the multiplex.</li>
</ol>
</li>
</ol>
2. General staining method
<ol>
<li>Preparation of wash and antigen retrieval solutions
<ol>
<li>Prepare the wash solution of 0.1% TBST by mixing 9 L of deionized water, 1 L of tris buffered saline (TBS) and 10 mL of polysorbate 20.</li>
<li>Prepare both antigen retrieval solutions (pH 6 and pH 9; Table of Materials) by diluting to 1x with deionized water.</li>
</ol>
</li>
<li>Deparaffinization and rehydration
<ol>
<li>Bake slides, lying flat, tissue side up at 60 &deg;C for 1 h in a hybridization oven1. Remove slides from the oven and allow to cool for at least 5&minus;10 min then place in a vertical slide rack.</li>
<li>Treat the slides in the following solutions sequentially: xylene in triplicate, followed by a single treatment of 100% ethanol, 95% ethanol, and lastly 70% ethanol for 10 min each using a slide staining set1.</li>
<li>Wash the rack of slides for 2 min with deionized water by submersion in a plastic slide box followed by fixation by submersion in a plastic slide box filled with neutral buffered formalin for 30 min13. Wash the slides in deionized water for 2 min then proceed to antigen retrieval.</li>
</ol>
</li>
<li>Antigen retrieval
<ol>
<li>Place the rack of slides into a heat resistant box filled to the internal line (approximately 300 mL) with pH 6 or pH 9 antigen retrieval buffer. NOTE: Antigen retrieval buffer can be either pH 6 or pH 9 which may need to be optimized per epitope. However, start by using pH 9 for antibodies that bind to nuclear epitopes and pH 6 for all other antibodies.</li>
<li>Cover the box with plastic wrap and use a rubber band to secure. Place the box into the microwave at the edge of the rotating plate (microwave must be equipped with inverter technology for even heating). Heat the slides for 45 s at 100% power followed by 15 min at 20% power13. NOTE: Microwave treatment may need optimization depending on the microwave used.</li>
<li>Let the slides cool for approximately 15&minus;20 min.</li>
</ol>
</li>
<li>Prepare working solutions of antibodies and fluorophores while slides cool.
<ol>
<li>Prepare the primary antibody diluent by dissolving 0.5 g of bovine serum albumin granules into 50 mL of TBST. Alternatively, use 50 mL of 1x phosphate buffered saline (PBS) to dissolve granules.</li>
<li>Make each primary antibody working solution to the optimized concentration determined in section 1, in the primary antibody diluent. Estimate approximately 200 &micro;L per slide.</li>
<li>Prepare the fluorophore working solution by diluting each fluorophore in the fluorophore diluent at a concentration of 1:100. NOTE: This may need to be optimized depending on the antibody. Estimate approximately 100 &micro;L per slide.</li>
<li>On the last day of staining, prepare the 4&prime;,6-diamidino-2-phenylindole (DAPI) working solution by adding 3 drops of DAPI into 1 mL of TBST.</li>
</ol>
</li>
<li>Slide staining (washing and blocking)
<ol>
<li>Remove the box from the microwave after cooling and take off the plastic wrap, wash the slides with deionized water for 2 min followed by a 2 min wash in a plastic slide box filled TBST13.</li>
<li>After drying each slide around the tissue with a delicate task wipe careful not to let the tissue dry out, trace around the outside of the tissue using a hydrophobic barrier pen. Do not touch the tissue with the delicate task wipe or pen.</li>
<li>Place each slide in the humidified chamber and add blocking solution (approximately 4 drops) to the tissue. Incubate for 10 min at room temperature (RT).</li>
</ol>
</li>
<li>Slide staining (primary antibody application)
<ol>
<li>Take each slide and remove the blocking solution by tapping the side of the slide on a stack of paper towels and using a delicate task wipe to remove excess blocking agent from around the tissue.</li>
<li>Lay the slide back into the humidified chamber and add approximately 200 &micro;L of the working primary antibody. Incubate in the humidified chamber for 1 h at RT.</li>
<li>Wash with TBST for 2 min by submersion in triplicate13. NOTE: After each washing step, changing the TBST will help ensure a cleaner image.</li>
</ol>
</li>
<li>Slide staining (secondary antibody application)
<ol>
<li>Dab off the remaining TBST from each slide and apply approximately 3 to 4 drops of secondary antibody (mixture of rabbit and mouse secondary HRP conjugated antibodies). Incubate for 10 min in the humidified chamber at RT13. NOTE: If planning to use a primary antibody that requires a secondary antibody other than mouse or rabbit or choosing to use an alternative secondary antibody, apply the appropriate HRP conjugated secondary to the slides and incubate at RT for 45 min. Optimization of incubation time may be needed for the alternative secondary14.</li>
<li>After incubation, wash with TBST for 2 min in triplicate13.</li>
</ol>
</li>
<li>Slide staining (fluorophore application)
<ol>
<li>Apply approximately 100 &micro;L of the fluorophore working solution and incubate in the humidified chamber at RT for 10 min13.</li>
<li>Wash with TBST for 2 min in triplicate13.</li>
</ol>
</li>
<li>Removal of antibodies
<ol>
<li>Microwave slides (45 s at 100% then 15 min at 20%) in the heat resistant box (see step 2.3.2) for removal of antibodies13,14. NOTE: This completes one round of the multiplex. This is the only point at which the protocol can be paused. To pause the protocol, leave the slides submerged in the antigen retrieval buffer overnight at room temperature.</li>
</ol>
</li>
<li>Continue additional rounds of staining. Repeat steps 2.5.1&minus;2.9.1 for the rest of the antibody-fluorophore pairs. Once all antibodies and fluorophores have been used, proceed to section 2.11.</li>
<li>DAPI application and mounting
<ol>
<li>After the last staining round in the multiplex, remove the last antibody application with antigen retrieval solution pH 613. Wash with deionized water followed by TBST for 2 min each13.</li>
<li>Apply approximately 150 &micro;L of the working DAPI solution to slides and incubate in the humidified chamber for 10 min at RT1.</li>
<li>Wash with TBST for approximately 30 s and mount coverslips with an antifade mountant. Apply clear fingernail polish at the four corners of the coverslip once the mounting media has dried to secure coverslip (optional).</li>
</ol>
</li>
</ol>
3. Details for library, monoplexes, and multiplex
<ol>
<li>Choose slides for building a fluorophore library.
<ol>
<li>For a 7-color multiplex gather 7 immune cell rich tissue slides (i.e., tonsil, spleen, etc.) for the library. NOTE: Choose control slides to be used for a fluorophore library with each slide representing each unique fluorophore that will be used in the multiplex. Control slides should have an abundant epitope of choice and may be the same type of tissue for each fluorophore.</li>
</ol>
</li>
<li>Stain and image library slides for use with monoplexes and multiplexes.
<ol>
<li>Follow steps 2.1&minus;2.9.1 using the control slides and control tissue slides of choice. Place a different fluorophore on each slide at a concentration of 1:100; one slide should contain only DAPI.</li>
<li>Proceed to section 2.11 except omit DAPI staining and instead use TBST and mount as described.</li>
<li>After letting slides dry overnight, image with all channels DAPI, CY3, CY5, CY7, Texas Red, semiconductor quantum dots, and fluorescein isothiocyanate (FITC) setting the exposure to 250 ms with the saturation protection feature1. NOTE: The exposure times can be set at 50&minus;250 ms13.</li>
<li>Upload the library images onto the software and use in the analysis of the monoplexes and multiplex.</li>
</ol>
</li>
<li>Choose slides for monoplexes.
<ol>
<li>Choose control slides that have an abundant epitope of choice based on optimized antibodies (section 1). For every round of staining to be done in the multiplex, select that number of control slides to create monoplexes. NOTE: The purpose of the monoplex is to determine the best position (order) for each antibody to be used in the multiplex.</li>
</ol>
</li>
<li>Stain monoplexes.
<ol>
<li>Follow sections 2.1&minus;2.11 using the primary antibody at a different order (position) for each of the slides. Each slide should have only one primary antibody applied at an order (position) different than the other slides. NOTE: For each slide where the round calls for no primary antibody or fluorophore, instead use primary antibody diluent and fluorophore diluent13.</li>
</ol>
</li>
<li>Image slides and analyze the monoplex.
<ol>
<li>Using the microscope and setting the exposure time to 250 ms for all channels capture each image utilizing DAPI to focus. NOTE: The exposure times can be set at 50&minus;250 ms14.</li>
<li>Using the analysis software evaluate each monoplex slide by looking at the fluorescent intensity of the stained marker.</li>
<li>Compare this intensity to the background. If it is at least 5&minus;10 times higher than the background, the position of that slide is an optimal position to use in the final multiplex.</li>
</ol>
</li>
<li>Choose slides for the multiplex.
<ol>
<li>Choose the slides of interest and add an extra slide to be used as a blank slide for subtraction of autofluorescence after staining and imaging.</li>
</ol>
</li>
<li>Stain the multiplex.
<ol>
<li>Based on the monoplex results, choose the appropriate order (positions) for each antibody based on step 3.5.3. Assign each fluorophore to an antibody. NOTE: This may need optimization; however, plan to use bright antibodies for less abundant markers1, co-localizing antibodies should be matched with fluorophores at far spectrums from each other13, and fluorophores with the spectrum 540 and 570 should not be co-localized due to spectral overlap issues1.</li>
<li>Proceed with sections 2.1&minus;2.11 ensuring the blank slide does not receive primary antibody, fluorophore, or DAPI, instead use antibody diluent, fluorophore diluent, and TBST respectively in its place.</li>
</ol>
</li>
<li>Image multiplex and analyze for integrity of staining
<ol>
<li>Using the microscope and setting the exposure time to 250 ms for all channels, capture each image utilizing DAPI to focus.</li>
<li>Using the analysis software (Table of Materials), evaluate each multiplex by fluorescent false color. NOTE: A clear image should demonstrate each marker clearly. If a marker looks diffuse and grainy or is nonexistent, it is likely that the marker didn&rsquo;t work and needs to be re-optimized.</li>
<li>Using the analysis software, evaluate each multiplex by pathology view. Pathology view will confirm the specificity of each marker. Compare to IHC previously optimized (section 1).</li>
</ol>
</li>
</ol>

<ol>
	<li>Lazarus, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+phenotypic+immune+profiling+of+metastatic+colon+cancer.">Spatial and phenotypic immune profiling of metastatic colon cancer.</a> JCI Insight. 3 (22), (2018).</li><li>Barua, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Functional+Spatial+Analysis+Platform+for+Discovery+of+Immunological+Interactions+Predictive+of+Low-Grade+to+High-Grade+Transition+of+Pancreatic+Intraductal+Papillary+Mucinous+Neoplasms.">A Functional Spatial Analysis Platform for Discovery of Immunological Interactions Predictive of Low-Grade to High-Grade Transition of Pancreatic Intraductal Papillary Mucinous Neoplasms.</a> Cancer Informatics. 17, 1176935118782880 (2018).</li><li>Yang, L., Liu, Z., Tan, J., Dong, H., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multispectral+imaging+reveals+hyper+active+TGF-beta+signaling+in+colorectal+cancer.">Multispectral imaging reveals hyper active TGF-beta signaling in colorectal cancer.</a> Cancer Biology &amp; Therapy. 19 (2), 105-112 (2018).</li><li>Carstens, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+computation+of+intratumoral+T+cells+correlates+with+survival+of+patients+with+pancreatic+cancer.">Spatial computation of intratumoral T cells correlates with survival of patients with pancreatic cancer.</a> Nature Communications. 8, 15095 (2017).</li><li>Steele, K. E., Brown, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+Immunohistochemistry+for+Image+Analysis+of+Tertiary+Lymphoid+Structures+in+Cancer.">Multiplex Immunohistochemistry for Image Analysis of Tertiary Lymphoid Structures in Cancer.</a> Methods In Molecular Biology. 1845, 87-98 (2018).</li><li>Gorris, M. A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eight-Color+Multiplex+Immunohistochemistry+for+Simultaneous+Detection+of+Multiple+Immune+Checkpoint+Molecules+within+the+Tumor+Microenvironment.">Eight-Color Multiplex Immunohistochemistry for Simultaneous Detection of Multiple Immune Checkpoint Molecules within the Tumor Microenvironment.</a> The Journal Of Immunology. 200 (1), 347-354 (2018).</li><li>Parra, E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+multiplex+immunofluorescence+panels+using+multispectral+microscopy+for+immune-profiling+of+formalin-fixed+and+paraffin-embedded+human+tumor+tissues.">Validation of multiplex immunofluorescence panels using multispectral microscopy for immune-profiling of formalin-fixed and paraffin-embedded human tumor tissues.</a> Scientific Reports. 7, (2017).</li><li>Katikireddy, K. R., O'Sullivan, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+and+immunofluorescence+procedures+for+protein+analysis.">Immunohistochemical and immunofluorescence procedures for protein analysis.</a> Methods In Molecular Biology. 784, 155-167 (2011).</li><li>Adan, A., Alizada, G., Kiraz, Y., Baran, Y., Nalbant, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry:+basic+principles+and+applications.">Flow cytometry: basic principles and applications.</a> Critical Reviews In Biotechnology. 37 (2), 163-176 (2017).</li><li>Zaritskaya, L., Shurin, M. R., Sayers, T. J., Malyguine, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+flow+cytometric+assays+for+monitoring+cell-mediated+cytotoxicity.">New flow cytometric assays for monitoring cell-mediated cytotoxicity.</a> Expert Review of Vaccines. 9 (6), 601-616 (2010).</li><li>Stack, E. C., Wang, C., Roman, K. A., Hoyt, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+immunohistochemistry,+imaging,+and+quantitation:+a+review,+with+an+assessment+of+Tyramide+signal+amplification,+multispectral+imaging+and+multiplex+analysis.">Multiplexed immunohistochemistry, imaging, and quantitation: a review, with an assessment of Tyramide signal amplification, multispectral imaging and multiplex analysis.</a> Methods. 70 (1), 46-58 (2014).</li><li>Zhang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fully+automated+5-plex+fluorescent+immunohistochemistry+with+tyramide+signal+amplification+and+same+species+antibodies.">Fully automated 5-plex fluorescent immunohistochemistry with tyramide signal amplification and same species antibodies.</a> Laboratory Investigation. 97 (7), 873-885 (2017).</li><li>Phenoptics Research Solutions. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Perkin Elmer Manual. , 32 (2016).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Opal Multiplex IHC Assay Development Guide. , (2014).</li><li>Carey, C. 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The authors have nothing to disclose.

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

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

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

optimization, design and avoiding pitfalls in manual multiplex fluorescent immunohistochemistry

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

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

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

Optimization, Design and Avoiding Pitfalls in Manual Multiplex Fluorescent Immunohistochemistry

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

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

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Research

JoVE Journal

Cancer Research

9.2K Views.  University of Michigan. 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.

Video: Optimization, Design and Avoiding Pitfalls in Manual Multiplex Fluorescent Immunohistochemistry

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 ...

Multiplex Immunofluorescence and Spatial Image Analysis of the Tumor Microenvironment

Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment

The tumor microenvironment (TME) is composed of a plethora of different cell types, such as cytotoxic immune cells and immunomodulatory cells. Depending on its composition and the interactions between cancer cells and peri-tumoral cells, the TME may affect cancer progression. The characterization of tumors and their complex microenvironment could improve the understanding of cancer diseases and may help scientists and clinicians to discover new biomarkers.
We recently developed several multiplex immunofluorescence (mIF) panels based on tyramide signal amplification (TSA) for the characterization of the TME in colorectal cancer, head and neck squamous cell carcinoma, melanoma, and lung cancer. Once the staining and scanning of the corresponding panels are completed, the samples are analyzed on an image analysis software. The spatial position and the staining of each cell are then exported from this quantification software into R. We developed R scripts that allow us not only to analyze the density of each cell type in several tumor compartments (e.g. the center of the tumor, the margin of the tumor, and the stroma) but also to perform distance-based analyses between different cell types.
This particular workflow adds a spatial dimension to the classical density analysis already routinely performed for several markers. mIF analysis could allow scientists to have a better understanding of the complex interaction between cancer cells and the TME and to discover new predictive biomarkers of response to treatments, such as immune checkpoint inhibitors, and targeted therapies.

Author Spotlight: Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment  

In this article, a protocol for manual tyramide signal amplification (TSA) multiplex immunofluorescence (mIF) combined with image analysis and spatial analysis is described. This protocol can be used with formalin-fixed paraffin-embedded (FFPE) sections for the staining of two to six antigens per slide depending on the slide scanner available in the laboratory.

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

Mapping the Tumor Immune Landscape: An In-Depth Multiplex IHC Analysis Protocol

Multiplex Immunohistochemical Analysis of the Spatial Immune Cell Landscape of the Tumor Microenvironment

The immune cell landscape of the tumor microenvironment potentially contains information for the discovery of prognostic and predictive biomarkers. Multiplex immunohistochemistry is a valuable tool to visualize and identify different types of immune cells in tumor tissues while retaining its spatial information. Here we provide detailed protocols to analyze lymphocyte, myeloid, and dendritic cell populations in tissue sections. Starting from cutting formalin-fixed paraffin-embedded sections, automatic multiplex staining procedures on an automated platform, scanning of the slides on a multispectral imaging microscope, to the analysis of images using an in-house-developed machine learning algorithm ImmuNet. These protocols can be applied to a variety of tumor specimens by simply switching tumor markers to analyze immune cells in different compartments of the sample (tumor versus invasive margin) and apply nearest-neighbor analysis. This analysis is not limited to tumor samples but can also be applied to other (non-)pathogenic tissues. Improvements to the equipment and workflow over the past few years have significantly shortened throughput times, which facilitates the future application of this procedure in the diagnostic setting.

Author Spotlight: Unlocking Insights into the Immune Cell Landscape of Tumors

This protocol describes in detail how immune cell characterization of the tumor microenvironment using multiplex immunohistochemistry is carried out.

The immune cell landscape of the tumor microenvironment potentially contains information for the discovery of prognostic and predictive biomarkers. ...

Deciphering Tumor Microenvironment Using mIHC in Lung Squamous Carcinoma

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

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 ...

In Situ Immunohistochemical Detection of Immune Cell Markers in Cancer Tissues

Multiplex Cyclic Fluorescent Immunohistochemistry

The tumor microenvironment involves interactions between host cells, tumor cells, immune cells, stromal cells, and vasculature. Characterizing and spatially organizing immune cell subsets and target proteins are crucial for prognostic and therapeutic purposes. This has led to the development of multiplexed immunohistochemistry staining methods. Multiplex fluorescence immunohistochemistry allows the simultaneous detection of multiple markers, facilitating a comprehensive understanding of cell function and intercellular interactions. In this paper, we describe a workflow for the multiplex cyclic fluorescent immunohistochemistry assay and its application in the quantification analysis of lymphocyte subsets. The multiplex cyclic fluorescent immunohistochemistry staining follows similar steps and reagents as standard immunohistochemistry, involving antigen retrieval, cyclic antibody incubation, and staining on a formalin-fixed paraffin-embedded (FFPE) tissue slide. During the antigen-antibody reaction, a mixture of antibodies from different species is prepared. Conditions, such as antigen retrieval time and antibody concentration, are optimized and validated to increase the signal-to-noise ratio. This technique is reproducible and serves as a valuable tool for immunotherapy research and clinical applications.

Author Spotlight: Efficient Detection of Immune Cell-Infiltration in Cancer Tissues Using Fluorescent Immunohistochemistry

Multiplex cyclic immunohistochemistry allows in situ detection of multiple markers simultaneously using repeated antigen-antibody incubation, image scanning, and image alignment and integration. Here, we present the operating protocol for identifying immune cell substrates with this technology in lung cancer and paired brain metastasis samples.

The tumor microenvironment involves interactions between host cells, tumor cells, immune cells, stromal cells, and vasculature. Characterizing and ...

Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic purposes. However, the visualization and quantification of multiple antigens in a single tissue section using conventional chromogenic IHC is challenging. Multiplexed imaging is especially relevant in lymphoma research and diagnostics, where markers have to be interpreted in the context of a complex tumor microenvironment. Here we describe a protocol for multiplexed fluorescent IHC staining to enable the quantitative assessment of multiple targets in specific cell types of interest in lymphoma.The method covers aspects of antibody validation, antibody optimization, the multiplex optimization with markers of lymphoma subtypes, the staining of tissue microarray (TMA) slides, and the scanning of the slides, followed by data analysis, with specific reference to lymphoma. Using this method, scores for both the mean intensity of a marker of interest and the percentage positivity are generated to facilitate further quantitative analysis. Multiplexing minimizes sample utilization and provides spatial information for each marker of interest.

Here we describe a protocol for multiplex fluorescent immunohistochemical staining and imaging for the simultaneous localization of multiple cancer-associated antigens in lymphoma. This protocol can be extended to the colocalization analysis of biomarkers within all tissue sections.

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic ...

Immunology and Infection

Visualization, Quantification, and Mapping of Immune Cell Populations in the Tumor Microenvironment

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density and the location of immune cells in the TME have important diagnostic and prognostic values. Multiomic profiling of the TME has exponentially increased our understanding of the numerous cellular and molecular networks regulating tumor initiation and progression. However, these techniques do not provide information about the spatial organization of cells or cell-cell interactions. Affordable, accessible, and easy to execute multiplexing techniques that allow spatial resolution of immune cells in tissue sections are needed to complement single cell-based high-throughput technologies. Here, we describe a strategy that integrates serial imaging, sequential labeling, and image alignment to generate virtual multiparameter slides of whole tissue sections. Virtual slides are subsequently analyzed in an automated fashion using user-defined protocols that enable identification, quantification, and mapping of cell populations of interest. The image analysis is done, in this case using the analysis modules Tissuealign, Author, and HISTOmap. We present an example where we applied this strategy successfully to one clinical specimen, maximizing the information that can be obtained from limited tissue samples and providing an unbiased view of the TME in the entire tissue section.

Here, we describe a simple and accessible strategy for visualizing, quantifying, and mapping immune cells in formalin-fixed paraffin-embedded tumor tissue sections. This methodology combines existing imaging and digital analysis techniques with the purpose of expanding the multiplexing capability and multiparameter analysis of imaging assays.

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density ...

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample that can provide information about antigen coexpression and spatial distribution of the markers. However, a lack of suitable antibodies for formalin-fixed tissues may restrict the nature of markers that can be detected. In addition, the staining method is time-consuming. Here we describe a rapid method to perform multispectral fluorescence imaging on frozen tissues. The method includes the fluorophore combinations used, detailed steps for the staining of mouse and human frozen tissues, and the scanning, acquisition, and analysis procedures. For staining analysis, a commercially available semiautomated multispectral fluorescence imaging system is used. Through this method, up to six different markers were stained and detected in a single frozen tissue section. The machine learning analysis software can phenotype cells that can be used for quantitative analysis. The method described here for frozen tissues is useful for the detection of markers that cannot be detected in FFPE tissues or for which antibodies are not available for FFPE tissues.

We describe a rapid staining method to perform multispectral imaging on frozen tissues.

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample ...

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of centromere-kinetochore proteins during the cell cycle remains a fundamental tool in investigating the mechanism(s) of these proteins. Advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores. In addition, methods of indirect immunofluorescent (IIF) staining using specific antibodies are crucial to these observations. However, despite numerous reports about IIF protocols, few discussed in detail problems of specific centromere-kinetochore proteins.1-4 Here we report optimized protocols to stain endogenous centromere-kinetochore proteins in human cells by using paraformaldehyde fixation and IIF staining. Furthermore, we report protocols to detect Flag-tagged exogenous CENP-A proteins in human cells subjected to acetone or methanol fixation. These methods are useful in detecting and quantifying endogenous centromere-kinetochore proteins and Flag-tagged CENP-A proteins, including those in human cells.

Here we report protocols to detect endogenous and exogenous centromere-kinetochore proteins in human cells and quantify these protein levels at centromeres-kinetochores by indirect immunofluorescent staining through the use of fixation (paraformaldehyde, acetone, or methanol fixation).

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of ...

Biology

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.