Name: Dual-modality Molecular Cartography: Integrating Multiplex mRNA Detection with Protein Imaging Mass Cytometry (Video) | JoVE Journal
Uploaded: 2025-11-14T12:45:00.000+00:00
Duration: 6 min 51 s
Description: Learn dual-modality molecular cartography for ovarian cancer research using multiplex mRNA detection with protein imaging mass cytometry. This protocol identifies therapeutic targets and biomarkers in FFPE tissue sections.

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5 minutes

This research was performed in collaboration with the Flow Cytometry and Cellular Imaging Core Facility, which is supported in part by the National Institutes of Health through M. D. Anderson&#39;s Cancer Center Support Grant P30 CA016672. This research was also supported in part by the MD Anderson&#39;s Cancer Center&#39;s Support Grant Development Award, Imaging Mass Cytometry Spatial Transcriptomics via RNAscope, P30CA016672, NIH/NCI, PI - Pisters. JKB effort was supported in part by the P30 CA016672 and the NCI Research Specialist 1 R50 CA243707-01A1. SFB effort was supported in part by the Ovarian Cancer Research Alliance (OCRA 811621 and 891490), The Honorable Tina Brozman Foundation for Ovarian Cancer Research, the MDACC Ovarian Cancer SPORE Developmental Research Program, NIH 1R01CA261952-01A1. SFB, JKB, and BG effort was supported in part by 1U01CA294459-01 grant. The authors would like to acknowledge Samuel Mok, who sectioned and provided the samples used.

This protocol uses formalin-fixed paraffin-embedded (FFPE) tissues collected from previously untreated patients undergoing primary cytoreductive surgery for high-grade serous ovarian carcinoma. All clinical data were obtained from the repository of the Department of Gynecologic Oncology and Reproductive Medicine under protocols approved by the University of Texas MD Anderson&#39;s Institutional Review Board. Written informed consent from the patients was obtained, and the studies were conducted in accordance with recognized ethical guidelines.
1. Precautions and preparations
<ol>
	<li>Always utilize nuclease free water (NFW) to prepare all needed reagents.</li>
	<li>Perform all steps using plastic containers and without the use of glass to reduce metal binding to glass and metal contamination.</li>
	<li>Keep the working area very clean and spray surfaces and pipettes with RNase decontamination solution, followed by 70% ethanol prepared with NFW.</li>
	<li>Conjugate thiolated mRNA detection probes (T1-T12) to proper metals via a malemide linker as described by Schulz et al7, further detailed in protocol.io in the Table of Materials. 
	NOTE: Protein targets should be chosen to indicate a cell&#39;s location within the tissue (structural markers), identify which cells are present (phenotypic markers), and what the cells are doing (functional markers). As some functional markers are transiently expressed, the inclusion of mRNA targets allows for the identification of cells generating these targets.</li>
</ol>
2. Sample preparation and preprocessing
<ol>
	<li>Bake tissue sections overnight in a dry oven at 60 &#176;C.</li>
	<li>Perform deparaffinization: Three rounds of Xylene for 5 min each round, and 10 rounds of rehydration (100% ethanol (EtOH) x2, 95% EtOH x2, 70% EtOH x2, 50% EtOH x2, 30% EtOH x2), for 3 min each round. Wash with 1x tris-buffered saline (TBS) for 2 min, once. According to how many samples are being processed at the same time, utilize 50 mL conical tubes (2 slides max) or 350 mL IHC staining cassettes (24 slides max). 
	NOTE: This dewaxing step, although recommended, is optional and dependent on tissue type.</li>
	<li>Apply a solution of 3.7% Paraformaldehyde (PFA) for 10 min by incubating the tissue slides in a 50 mL tube with enough PFA solution to cover the tissue section.</li>
	<li>Heat Induced Antigen Retrieval (HEIR) using microwave: Using a temperature-monitored microwave, fill one tray with pH 6 HEIR Antigen retrieval buffer (350 mL) and the remaining trays with NFW. Place tissue sections into a staining rack for microscope slides and add them to the tray with antigen retrieval buffer. 
	NOTE: When performing antigen retrieval, ensure the antigen retrieval buffer is at room temperature (RT) before use.</li>
	<li>Perform antigen retrieval at 107 &#176;C for 15 min, then let tissue slides cool down at RT for 20 min in a chemical hood.</li>
	<li>During antigen retrieval, rinse humidifying paper in water, place it in a humidity control slide staining tray, and place the tray in a hybridization oven at 60 &#176;C.</li>
	<li>Rinse slides in RNase-free TBS for 3 min, then dry slides for 3 min.</li>
</ol>
3. Protease treatment
<ol>
	<li>Draw a hydrophobic barrier around each tissue section with a hydrophobic barrier pen. Let the barrier dry completely for approximately 2 min.</li>
	<li>Load the dry slides into the humidity tray&#39;s slide holder by opening the swing clamp.</li>
	<li>Add enough drops of protease to entirely cover the tissue section. Remove the pre-warmed humidity control tray (60 &#176;C, step 2.6) from the hybridization oven and insert the slide holder into the humidity control tray. Re-seal the humidity control tray and return it to the hybridization oven. Incubate at 40 &#176;C for 30 min.</li>
	<li>During this incubation, prepare RNA-ISH Assay materials.
	<ol>
		<li>Prepare 500 mL of 1x RNA-ISH wash buffer by diluting the 50x stock with NFW. Store at RT up to one month. 
		NOTE: If precipitation occurs in the 50x wash buffer, resuspend by heating the buffer to 40 &#176;C for 10-20 min prior to preparing the 1x wash buffer.</li>
	</ol>
	</li>
	<li>Prepare 1 L of DTBS-T (0.5% Tween) in NFW by adding 5 mL of Tween to 1 L of DPBS-T.</li>
	<li>Prepare RNA-ISH ZZ-paired Target Probes:
	<ol>
		<li>Warm mRNA probe stocks and RNA-ISH diluent at 40 &#176;C in a dry oven for 10 min. Vortex the probes and spin them down in a benchtop mini centrifuge.</li>
		<li>Prepare 1x mix (up to 12 probes, Table 1) by diluting stock 50x probes in RNA-ISH diluent, making sure to have enough final volume to cover the entire tissue section.</li>
		<li>Vortex well and leave the mixed probes and RNA-ISH diluent at RT until use.</li>
	</ol>
	</li>
	<li>Remove the humidity control tray from the hybridization oven and remove the slide holder. Return the tray to the oven.</li>
	<li>Place the slides into a clear slide holder pre-washed with NFW. Wash the slides in NFW with slight agitation for 5 min.</li>
	<li>Repeat the wash step with fresh NFW for 5 min.</li>
</ol>
4. Target probes hybridization
<ol>
	<li>Remove excess liquid from the slides. Remove the humidity control tray from the oven and place the slide holder into the tray.</li>
	<li>Add enough of the appropriate probe mix to entirely cover each tissue section. Close the tray and insert it into the oven for 2 h at 40 &#176;C.</li>
	<li>Place RNA-ISH Amplifier 1 (ZZ tail trees for tails T1 to T4) reagent to equilibrate at RT 30 min before the end of the incubation, to use in step 5.2.</li>
	<li>Once incubation is done, remove the humidity control tray from the hybridization oven and remove the slide holder. Return the tray to the oven.</li>
	<li>Place the slides into a clear slide holder pre-washed with NFW. Wash the slides in 1x wash buffer with slight agitation for 2 min.</li>
	<li>Repeat the wash step with fresh 1x Wash Buffer for 2 min at RT.</li>
</ol>
5. Hybridize RNA-ISH amplifier 1
<ol>
	<li>Remove excess liquid from the slides and place the slides in the humidity tray&#39;s slide holder. Insert the slide holder into the humidity control tray.</li>
	<li>Vortex RNA-ISH Amplifier 1 and add it to entirely cover each section. Close the tray and insert it into the hybridization oven for 30 min at 40 &#176;C. Place RNA-ISH Amplifier 2 (ZZ tail trees for tails T5 to T8) reagent at RT, to use in step 6.2.</li>
	<li>Remove the humidity control tray from the hybridization oven and remove the slide holder. Return the tray to the oven.</li>
	<li>Place the slides into a clear slide holder pre-washed with NFW. Wash the slides in 1x wash buffer with slight agitation for 2 min.</li>
	<li>Repeat the wash step with fresh 1x wash buffer for 2 min at RT.</li>
</ol>
6. Hybridize RNA-ISH amplifier 2
<ol>
	<li>Remove excess liquid from the slides. Remove the humidity control tray from the oven and place the slide holder into the tray</li>
	<li>Vortex RNA-ISH Amplifier 2 and add it to cover each section. Close the tray and insert it into the hybridization oven for 30 min at 40 &#176;C.</li>
	<li>Place RNA-ISH Amplifier 3 (ZZ tail trees for tails T9 to T 12) reagent at RT, to use in step 7.2. Remove the slide holder from the tray. Place the tray back into the oven.</li>
	<li>Place the slides into a clear slide holder pre-washed with NFW. Wash the slides in 1x wash buffer with slight agitation for 2 min.</li>
	<li>Repeat the wash step with fresh 1x wash buffer for 2 min at RT.</li>
</ol>
7. Hybridize RNA-ISH amplifier 3
<ol>
	<li>Remove excess liquid from the slides. Remove the humidity control tray from the oven and place the slide holder into the tray</li>
	<li>Vortex RNA-ISH Amplifier 3 and add it to entirely cover each section. Close the tray and insert it into the hybridization oven for 30 min at 40 &#176;C.</li>
	<li>During incubation, prepare the metal oligos mix for step 8. Each oligo is used at a concentration of 1:15 (from 10uM stock, making sure to have enough final volume to cover the entire tissue section). Vortex well and leave the mixed metal oligos at RT until use. Take the humidity control tray out of the oven, then remove the slide holder from the tray. Afterward, return the tray to the oven</li>
	<li>Place the slides into a clear slide holder pre-washed with NFW. Wash the slides in 1x wash buffer with slight agitation for 2 min.</li>
	<li>Repeat the wash step with fresh 1x Wash Buffer for 2 min at RT.</li>
</ol>
8. Hybridize metal oligos
<ol>
	<li>Remove excess liquid from the slides. Remove the humidity control tray from the oven and place the slide holder into the tray. Add mixed metal oligos prepared in step 7.3 (vortex before use).</li>
	<li>Close the tray and insert it into the hybridization oven for 45 min at 40 &#176;C.</li>
	<li>Remove the humidity control tray from the hybridization oven and remove the slide holder. Return the tray to the oven.</li>
	<li>Place the slides into a clear slide holder pre-washed with NFW. Wash the slides in 1x wash buffer with slight agitation for 2 min.</li>
	<li>Repeat the wash step with fresh 1x Wash Buffer for 2 min at RT.</li>
</ol>
9. Metal conjugated antibodies
<ol>
	<li>To block non-specific sites, apply enough volume of blocking buffer to cover the entire tissue section and incubate for 30 min at RT.</li>
	<li>During incubation, prepare the antibody mix to be used in step 10.1 by diluting the antibodies (Table 1) in Antibody diluent buffer.</li>
</ol>
10. Incubation with metal conjugated antibodies
<ol>
	<li>After blocking buffer incubation, discard the blocking buffer from the tissue slides and dispense enough volume of the Ab mix (from step 9.2) to cover the entire tissue section.</li>
	<li>Place the slide in a humidity chamber and incubate overnight (~16 h) at 4 &#176;C.</li>
</ol>
11. DNA staining
<ol>
	<li>Prepare fresh Ir-Intercalator solution.
	<ol>
		<li>From a 500 &#181;M stock solution, prepare a 1:2000 working solution in RNase-free TBS. Working solution can be aliquoted and stored at -20 &#176;C (do not freeze-thaw).</li>
	</ol>
	</li>
	<li>Place the slides into a clear slide holder pre-washed with NFW. Wash the slides in TBS-T buffer with slight agitation for 5 min.</li>
	<li>Repeat wash in TBS-T.</li>
	<li>Stain slides with Ir working solution (from step 11.1.1) for 5 min at RT.</li>
	<li>Place the slides into a clear slide holder pre-washed with NFW. Wash the slides in TBS-T buffer with slight agitation for 5 min.</li>
	<li>Repeat wash in TBS-T.</li>
	<li>Wash twice in TBS, 5 min (stir slowly).</li>
	<li>Dip the slide quickly in ddH2O to avoid crystallization of the salts present in the TBS on the tissue.</li>
	<li>Dry the slide under a chemical hood (10 min) and store at 4 &#176;C until image acquisition.</li>
</ol>
12. Image acquisition
<ol>
	<li>Load the slide into the ablation chamber of IMC.</li>
	<li>Capture a panorama image to identify the region of interest (ROI).</li>
	<li>Map out ROIs in the image acquisition software.</li>
	<li>Apply the acquisition template.</li>
	<li>Adjust laser power (ablation energy) based on the sample type. The default is set at 0 dB. 
	NOTE: It is advised to test the ablation energy with multiple small tissue areas (30 &#181;m x 30 &#181;m), increasing the power by 1 dB each time. Select the lowest ablation energy that clears the specimen without burning into the slide. Normally, this results in a laser power of 3-4 dB. Occasionally, if the tissue is thicker than advised, the laser power will increase to 5 dB.</li>
	<li>Initiate laser ablation. The system directs the laser to the ROI and ablates the metal-tagged proteins, generating aerosol plumes that are transferred to the inductively coupled plasma (ICP) Torch, where the plumes are vaporized, atomized, and ionized in plasma.</li>
	<li>Measure the quantity of each isotope that is measured by the detector and converted into data.</li>
</ol>

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<li>Rivest, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fully+automated+sequential+immunofluorescence+%28seqIF%29+for+hyperplex+spatial+proteomics%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Fully automated sequential immunofluorescence (seqIF) for hyperplex spatial proteomics.</a> Sci Rep. 13 (1), 1-12 (2023).</li>
<li>Sans, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spatial+transcriptomics+of+intraductal+papillary+mucinous+neoplasms+of+the+pancreas+identifies+NKX6-2+as+a+driver+of+gastric+differentiation+and+indolent+biological+potential%5BTitle%5D)+AND+%22Cancer+Discov%22%5BJournal%5D)">Spatial transcriptomics of intraductal papillary mucinous neoplasms of the pancreas identifies NKX6-2 as a driver of gastric differentiation and indolent biological potential.</a> Cancer Discov. 13 (8), 1844-1861 (2023).</li>
<li>Glasson, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+high-dimensional+imaging+mass+cytometry%3A+one+step+beyond+in+oncology%5BTitle%5D)+AND+%22Semin+Immunopathol%22%5BJournal%5D)">Single-cell high-dimensional imaging mass cytometry: one step beyond in oncology.</a> Semin Immunopathol. 46 (1), 1-12 (2023).</li>
</ol>

Jared K. Burks is a consultant for Standard BioTools.

Multiplex multi-omics is a critical evolution in the data collected, targeting cell-to-cell and cell-to-neighborhood interactions that impact disease progression and treatment. Analyzing this data in the same sample and thus the same cell is critical, as many protein targets are transiently expressed, or differ in mRNA and protein expression level, thus bringing in a targeted mRNA detection combined with protein allows for accommodation of difficult to detect or differentially expressing targets21...

Cancers exhibit structural and functional diversities in various patients. The tumor microenvironment (TME), composed of normal and malignant cells, is heterogeneous among patients. Metastasis results from a residue from the primary tumor escaping into the bloodstream as cell clusters and single cells, which provides clues about disease progression and therapeutic response1,2. The complexity of these microenvironments needs to be understood1. Although tumors comprise ample cell types, the clinical standard is histologically based on a single marker, which is starting to change1,2. Multiplexed imaging technologies have afforded new directions in pathology. Spatially resolved proteomics, genomics, metabolomics, and lipidomics of human cancers are now possible at or near single cell resolution3,4,5,6.
These technologies can dissect the heterogeneous cellular locations and interactions in tumors. Utilizing the appropriate single-cell bioimaging methodology allows for the generation of profiles of many disease-associated protein biomarkers in patient biopsies to inform the design of cancer therapies. Insights from spatial cellular maps will be able to guide the choice for combination therapy that can efficiently eliminate cancers with reduced off-targets, resistance, and relapse1. At issue is the ability to unionize these many varied technologies that typically do not work in harmony on the same section of tissue7. This protocol, visually outlined in Figure 1, offers the opportunity to spatially examine both proteomics and a targeted subset of mRNAs in the same tissue section and the same cell simultaneously at a higher plexity than what is possible with simultaneous fluorescent protein+RNA-ISH8. This can also serve as a bridge for parallel analysis3,4,6, with the goal of deconvoluting the analysis of the data. While dual detection is present, one can also examine cells that have rapidly initiated mRNA production, but for which the secreted protein remains undetectable due to the limited sensitivity and lack of signal amplification inherent to directly labeled primary antibody methods, making the detection of secreted molecules particularly challenging. The following protocol is adapted from a 12-plex fluorescent/chromogenic detection to be applicable for metal-based detection via thiol-reduced modified probes for simultaneous detection in IMC.

<table><tbody><tr><td>&amp;nbsp;https://www.protocols.io/view/oligonucleotide-polymer-conjugation-for-imaging-ma-dm6gpjz91gzp/v2.</td><td>Protocols.IO</td><td>dx.doi.org/10.17504/protocols.io.dm6gpjz91gzp/v2</td><td>Oligonucleotide-polymer conjugation for imaging mass cytometry V.2&amp;nbsp;</td></tr><tr><td>10x TBS RNAse-Free</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>J62780.K7</td><td>For intermittent washes between steps</td></tr><tr><td>ACD HybEZ II Hybridization System</td><td>ACD Biotechne</td><td>321711</td><td>Oven for baking tissue slides</td></tr><tr><td>Antibody Diluent</td><td>Dako</td><td>S0809&amp;nbsp;</td><td>Antibody diluent buffer</td></tr><tr><td>Antibody: B7-H3</td><td>Bethyl</td><td>A700-025CF, A700-026CF</td><td>Clone: BLR025F, BLR026F</td></tr><tr><td>Antibody: B7-H4</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>14-5949-82</td><td>Clone: H74</td></tr><tr><td>Antibody: BRD4(6F11)</td><td>Bethyl</td><td>A700-005CF</td><td>Clone: BL-151-6F11</td></tr><tr><td>Antibody: CCR6</td><td>R&amp;amp;D</td><td>MAB195-100</td><td>Clone: 53103</td></tr><tr><td>Antibody: CD11b</td><td>Abcam&amp;nbsp;</td><td>ab209970</td><td>Clone: EPR1344</td></tr><tr><td>Antibody: CD25</td><td>Abcam&amp;nbsp;</td><td>ab215378</td><td>Clone: EPR6452</td></tr><tr><td>Antibody: CD278/ICOS</td><td>CST&amp;nbsp;</td><td>89601BF</td><td>Clone: D1K2T</td></tr><tr><td>Antibody: CD31</td><td>Abcam&amp;nbsp;</td><td>ab207090</td><td>Clone: EPR3094</td></tr><tr><td>Antibody: CD33</td><td>Abcam&amp;nbsp;</td><td>ab238784</td><td>Clone: SP266</td></tr><tr><td>Antibody: CD3e&amp;nbsp;</td><td>CST</td><td>85061BF</td><td>Clone: D7A6E</td></tr><tr><td>Antibody: CD4</td><td>Abcam</td><td>ab181724</td><td>Clone: EPR6855</td></tr><tr><td>Antibody: CD44</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>MA1-10225</td><td>Clone: IM7</td></tr><tr><td>Antibody: CD45</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>14-9457-82</td><td>Clone: CD45-2B11</td></tr><tr><td>Antibody: CD8a</td><td>CST&amp;nbsp;</td><td>85336BF</td><td>Clone: D8A8Y</td></tr><tr><td>Antibody: CXCL13</td><td>R&amp;amp;D</td><td>AF801</td><td>Clone: Polyclonal</td></tr><tr><td>Antibody: CXCR5</td><td>R&amp;amp;D</td><td>MAB190-100</td><td>Clone: CXCR5</td></tr><tr><td>Antibody: gamma-H2AX</td><td>Bethyl</td><td>A700-053CF</td><td>Clone: BLR053F</td></tr><tr><td>Antibody: Granzyme B</td><td>Santa Cruz</td><td>sc-8022 x</td><td>Clone: 2C5</td></tr><tr><td>Antibody: KAP1</td><td>Bethyl</td><td>A700-014CF</td><td>Clone: BL-248-2G6</td></tr><tr><td>Antibody: Ki67</td><td>Bethyl</td><td>A700-021CF</td><td>Clone: BLR021E</td></tr><tr><td>Antibody: LCK&amp;nbsp;</td><td>CST</td><td>2984BF</td><td>Clone: D88</td></tr><tr><td>Antibody: MFAP5</td><td>N/A</td><td>N/A</td><td>PMID: 31332047 / Clone: 130A&amp;nbsp; (Note: custom designed)&amp;nbsp;</td></tr><tr><td>Antibody: SMA</td><td>CST</td><td>56856BF</td><td>Clone: 1A4</td></tr><tr><td>Antibody: VEGF</td><td>STB-Fluidigm</td><td>3163028D</td><td>Clone: G153-494</td></tr><tr><td>Antibody: VISTA</td><td>CST</td><td>64953BF</td><td>Clone: D1L2G</td></tr><tr><td>AZ-AR1 Elegance Citra&amp;nbsp;</td><td>BioGeneX</td><td>HK546-XAK</td><td>Antigen retrieval buffer</td></tr><tr><td>Ethyl Alcohol 100% (200 Proof)</td><td>Pharmco</td><td>111000200</td><td>For deparaffinization</td></tr><tr><td>EZ &amp;ndash; Retriever IR</td><td>BioGeneX</td><td>MW016-IR</td><td>Antigen Retrieval System for FFPE Tissue Sections</td></tr><tr><td>HybEZ II Hybridization System</td><td>ACD Biotechne</td><td>321710</td><td>The system comprises: HybEZ oven (PN 321710/321720), a humidity control tray (PN 310012), and HybEZ Humidifying Paper (2 sheets PN 310025), EZ-Batch Wash Tray (PN 321717), EZ-Batch Slide Holder (PN 321716)</td></tr><tr><td>ImmEdge Hydrophobic Barrier PAP Pen&amp;nbsp;</td><td>VectorLabs</td><td>H-4000</td><td>Hydrophobic Barrier Pen</td></tr><tr><td>Ir Intercalator Solution</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>NC1038184</td><td>Used for identifying nucleated cells</td></tr><tr><td>KERATIN 8/18</td><td>CST&amp;nbsp;</td><td>4546BF</td><td>Clone: C51</td></tr><tr><td>Metal Oligos</td><td>Various</td><td>Various</td><td>Metal tag ranging from T1 to T12 depending on which is needed</td></tr><tr><td>Nuclease-free water (NFW)</td><td>Corning&amp;nbsp;</td><td>46-000-CM&amp;nbsp;</td><td>To clean and prep all equipment, and to use as a dilutant when necessary</td></tr><tr><td>RNAscope Protease Plus&amp;nbsp;</td><td>ACD Biotechne</td><td>322331&amp;nbsp;</td><td>Used to permeabilize cell membranes of samples</td></tr><tr><td>RNAscope EZ-Batch Slide Holder</td><td>ACD Biotechne</td><td>310017</td><td>Designed to manually batch process up to 20 slides efficiently</td></tr><tr><td>RNAscope HiPlex12 Reagents Kit v2&amp;nbsp;</td><td>ACD Biotechne</td><td>324409</td><td>Reagents for RNAScope</td></tr><tr><td>RNAscope HiPlex Probe - Target Probes</td><td>ACD Biotechne</td><td>Various</td><td>Range from T1 to T12 dependent on which is needed</td></tr><tr><td>RNaseZap RNase Decontamination Solution</td><td>Thermo Fisher Scientific</td><td>AM9782</td><td>A surface decontamination solution that destroys RNases</td></tr><tr><td>Staining Rack Microscope Slide Basket Holder 24 Place Chemical Resistant Plastic</td><td>Optimal Scientific</td><td>3028</td><td>For staining multiple slides</td></tr><tr><td>Tween 20</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>AAJ20605AP</td><td>For intermittent washes between steps</td></tr><tr><td>Ultravision Protein Block</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>TA125PBQ</td><td>Blocking buffer</td></tr></tbody></table>

dual-modality molecular cartography: integrating multiplex mrna detection with protein imaging mass cytometry

Dual-modality Molecular Cartography: Integrating Multiplex mRNA Detection with Protein Imaging Mass Cytometry

The ability to detect protein and mRNA in the same assay helps us understand how the cell is priming for an arrival and what cues it receives as it travels and interacts in the environment it finds itself in. Higher-plexed imaging now permits characterization of spatially resolved mRNA (spatial transcriptomics) and protein (spatial proteomics) simultaneously. To avoid redundancy, these analytes can be analyzed in a single tissue section. There are many technologies that allow for many proteins and few mRNAs or the inverse, but as the central acting analytes are proteins, this manuscript is focused on integrating protein imaging mass cytometry (IMC) with mRNA in situ hybridization (ISH) utilizing metal probes. This allows us to understand what messages the cell is priming or is sending rapidly into the world as it communicates in its microenvironment. It also overcomes limitations related to the detection of secreted proteins or challenging markers that are usually hard to quantify and visualize utilizing antibodies only. This protocol also minimizes the need for compensations or post-processing seen in higher plex fluorescent-based methods, as there is no fluorescent spectral spillover in a metal probe-based detection system, nor are there tissue-specific autofluorescence considerations.

The ability to detect protein and mRNA in the same assay helps us understand how the cell is priming for an arrival, but also what queues it is receiving as it travels and interacts with its environment. As seen in the representative data, mRNA and protein detection are critical components in spatial biology research, as the lower limits of detection can be quite challenging based on the way the labeling, detector, and/or imaging steps progress. Just the inclusion of proteases can complicate critical markers in combined ...

This protocol details a methodology for multiplex mRNA detection with protein imaging mass cytometry in formalin fixed paraffin embedded tissue sections.

The ability to detect protein and mRNA in the same assay helps us understand how the cell is priming for an arrival and what cues it receives as it ...

We focus on special biology to further understand the molecular mechanisms of ovarian cancer in the hopes to find new therapeutical targets and biomarkers to predict patient survival. One of the current IMC challenges includes dissection of lower abundance protein, the labor of antibody validation and titration, as well as the complex and time-consuming pixel classification during analysis. Begin by removing excess liquid from the prepared FFPE slides.Take the humidity control tray out of the oven, and place the slide holder into the tray. Then, vortex RNA-ISH Amplifier 3 thoroughly, and add enough reagent to completely cover each tissue section on the slides. Close the tray, and insert it into the hybridization oven for 30 minutes at 40 degrees Celsius.During incubation, prepare the metal oligonucleotide mix by diluting each oligo at a one-to-15 ratio. Vortex the metal oligo mix thoroughly, and leave it at room temperature until further use. Now, remove the humidity control tray from the oven.Take the slide holder out of the tray, and place the tray back into the oven. Then, place the slides into a clear slide holder that has been pre-washed with nuclease-free water. Wash the slides in wash buffer under slight agitation for two minutes.After removing excess liquid from the slides, take the humidity control tray out of the oven, and place the slide holder into the tray. Add the mixed metal oligonucleotides prepared earlier to fully cover each tissue section. Now, close the tray completely, and insert it into the hybridization oven for 45 minutes at 40 degrees Celsius.Remove the humidity control tray from the hybridization oven, take out the slide holder, and return the empty tray to the oven. To block nonspecific binding sites, add a sufficient volume of blocking buffer to fully cover each tissue section on the slide, and incubate the slides at room temperature for 30 minutes. While the slides are incubating, prepare the antibody mix by diluting the antibodies using the antibody diluent buffer.Once the incubation is over, discard the buffer from the tissue slides, and dispense enough of the previously prepared antibody mix to completely cover the tissue sections. Then, place the slides into a humidity chamber, and incubate them overnight at four degrees Celsius. To prepare a fresh Iridium intercalator solution, make a one-to-2, 000 dilution from a 500-micromolar stock solution using RNase-free TBS.Place the slides into a clear slide holder that has been pre-washed with nuclease-free water, and wash the slides in TBS-T buffer using slight agitation for five minutes. Then, to stain the slides, add the prepared Iridium working solution, and incubate for five minutes at room temperature. After that, place the slides into a clear slide holder that has been pre-washed with nuclease-free water.Wash the slides two times in TBS-T buffer with slight agitation for five minutes. Then, dip the slides quickly in double-distilled water to prevent salt crystallization from TBS on the tissue. Dry the slides under a chemical hood for 10 minutes, and store them at four degrees Celsius until image acquisition.Load the prepared slide into the ablation chamber of the imaging mass cytometry system. Capture a panorama image of the slide to identify the region of interest. Using the image acquisition software, mark the desired regions of interest on the panorama image.Apply the acquisition template in the image acquisition software. And adjust the laser power based on the sample type. Now, start the laser ablation process.Measure the quantity of each isotope using the detector, which converts the data into digital form for analysis. The activation status of CD8 T cells was assessed using co-detection of granzyme B at both mRNA and protein levels, along with interferon-gamma positivity. A cancer-associated fibroblast was shown to express MFAP5 protein and POSTN mRNA, both markers of cancer-associated fibroblast aggressiveness.A CD4 T cell was shown to co-express IL17A and IL6 mRNA, consistent with TH17 cell identity. CD4 T cells were observed expressing IL13 mRNA. Keratin-positive CD47-positive tumor cells were shown near a CD8 T cell.An activated CD8 T cell was shown to express CD25 protein. Comparable mRNA staining of THBS2 and CD47 was observed between metal-conjugated and fluorescent-labeled protocols.

dual-modality-molecular-cartography-integrating-multiplex-mrna

Research

JoVE Journal

Cancer Research

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.

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

We demonstrate a method to image multiple molecules within heterogeneous nano-structures at single molecule accuracy using sequential binding and elution of fluorescently labeled antibodies.

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

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

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

Here, we describe a protocol for fine-tuning regions of interest (ROIs) for Spatial Omics technologies to better characterize the tumor microenvironment and identify specific cell populations. For proteomics assays, automated customized protocols can guide ROI selection, while transcriptomics assays can be fine-tuned utilizing ROIs as small as 50 µm.

Spatial Profiling of Protein and RNA Expression in Tissue: An Approach to Fine-Tune Virtual Microdissection

In the era of cancer immunotherapy, interest in elucidating tumor microenvironment dynamics has increased strikingly. This protocol details a mass spectrometry imaging technique with respect to its staining and imaging steps, which allow for highly multiplexed spatial analysis.

Expanding the Comprehension of the Tumor Microenvironment using Mass Spectrometry Imaging of Formalin-Fixed and Paraffin-Embedded Tissue Samples

Multiplexed barcoding image analysis has recently improved the characterization of the tumor microenvironment, permitting comprehensive studies of cell composition, functional state, and cell-cell interactions. Herein, we describe a staining and imaging protocol using the barcoding of oligonucleotide-conjugated antibodies and cycle imaging, which allows for the use of a high-dimensional image analysis technique.

Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples

This protocol outlines a comprehensive mass cytometry (cytometry by time-of-flight [CyTOF]) analysis method for evaluating both systemic and local immune responses in hepatocellular carcinoma (HCC). The approach aims to provide insights into the immune landscape of HCC, offering a deeper understanding of the tumor microenvironment and the associated immune mechanisms.

Mass Cytometry Analysis of Systemic and Local Immune Responses in Hepatocellular Carcinoma

This report describes an automated protocol for multiplex immunofluorescence (mIF) assays on an automated slide stainer. It demonstrates the high reproducibility of spatial proteomics on standard high-throughput tissue autostainers and whole-slide fluorescence imagers, which is ideal for custom mIF assays across a large number of tissue slides in translational research.

High-Throughput Automated Multiplex Immunofluorescence Assays for Translational Research

This protocol provides a step-by-step guide for designing a multiplex immunofluorescence antibody panel for DNA-barcode-based imaging of murine FFPE melanoma tissues. We also describe an image analysis pipeline using open-source tools for generating spatial proteomics insights into the murine melanoma tumor immune microenvironment.

DNA-barcode-based Multiplex Immunofluorescence Imaging to Analyze FFPE Specimens from Genetically Reprogrammed Murine Melanoma

Orthotopic intracranial injection of tumor cells has been used in cancer research to study brain tumor biology, progression, evolution, and therapeutic response. Here we present fluorescence molecular tomography of tumor xenografts, which provides real-time intravital imaging and quantification of a tumor mass in preclinical glioblastoma models.

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Learn dual-modality molecular cartography for ovarian cancer research using multiplex mRNA detection with protein imaging mass cytometry. This protocol identifies therapeutic targets and biomarkers in FFPE tissue sections.

Dual-modality Molecular Cartography: Integrating Multiplex mRNA Detection with Protein Imaging Mass Cytometry

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