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Method Article

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

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DOI:

10.3791/68616

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November 14th, 2025

 ,  ,  ,  ,  ,  ,  ,  ,  ,  ,  , 
1Department of Gynecologic Oncology and Reproductive Medicine, The University of Texas MD Anderson Cancer Center, 2Department of Hematopoietic Biology & Malignancies, The University of Texas MD Anderson Cancer Center, 3Department of Leukemia, The University of Texas MD Anderson Cancer Center

In This Article

Summary

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

Abstract

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.

Introduction

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.

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Protocol

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'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

  1. Always utilize nuclease free water (NFW) to prepare all needed reagents.
  2. Perform all steps using plastic containers and without the use of glass to reduce metal binding to glass and metal contamination.
  3. Keep the working area very clean and spray surfaces and pipettes with RNase decontamination solution, followed by 70% ethanol prepared with NFW.
  4. 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'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.

2. Sample preparation and preprocessing

  1. Bake tissue sections overnight in a dry oven at 60 °C.
  2. 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.
  3. 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.
  4. 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.
  5. Perform antigen retrieval at 107 °C for 15 min, then let tissue slides cool down at RT for 20 min in a chemical hood.
  6. 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 °C.
  7. Rinse slides in RNase-free TBS for 3 min, then dry slides for 3 min.

3. Protease treatment

  1. Draw a hydrophobic barrier around each tissue section with a hydrophobic barrier pen. Let the barrier dry completely for approximately 2 min.
  2. Load the dry slides into the humidity tray's slide holder by opening the swing clamp.
  3. Add enough drops of protease to entirely cover the tissue section. Remove the pre-warmed humidity control tray (60 °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 °C for 30 min.
  4. During this incubation, prepare RNA-ISH Assay materials.
    1. 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 °C for 10-20 min prior to preparing the 1x wash buffer.
  5. Prepare 1 L of DTBS-T (0.5% Tween) in NFW by adding 5 mL of Tween to 1 L of DPBS-T.
  6. Prepare RNA-ISH ZZ-paired Target Probes:
    1. Warm mRNA probe stocks and RNA-ISH diluent at 40 °C in a dry oven for 10 min. Vortex the probes and spin them down in a benchtop mini centrifuge.
    2. 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.
    3. Vortex well and leave the mixed probes and RNA-ISH diluent at RT until use.
  7. Remove the humidity control tray from the hybridization oven and remove the slide holder. Return the tray to the oven.
  8. Place the slides into a clear slide holder pre-washed with NFW. Wash the slides in NFW with slight agitation for 5 min.
  9. Repeat the wash step with fresh NFW for 5 min.

4. Target probes hybridization

  1. Remove excess liquid from the slides. Remove the humidity control tray from the oven and place the slide holder into the tray.
  2. 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 °C.
  3. 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.
  4. Once incubation is done, remove the humidity control tray from the hybridization oven and remove the slide holder. Return the tray to the oven.
  5. 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.
  6. Repeat the wash step with fresh 1x Wash Buffer for 2 min at RT.

5. Hybridize RNA-ISH amplifier 1

  1. Remove excess liquid from the slides and place the slides in the humidity tray's slide holder. Insert the slide holder into the humidity control tray.
  2. 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 °C. Place RNA-ISH Amplifier 2 (ZZ tail trees for tails T5 to T8) reagent at RT, to use in step 6.2.
  3. Remove the humidity control tray from the hybridization oven and remove the slide holder. Return the tray to the oven.
  4. 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.
  5. Repeat the wash step with fresh 1x wash buffer for 2 min at RT.

6. Hybridize RNA-ISH amplifier 2

  1. Remove excess liquid from the slides. Remove the humidity control tray from the oven and place the slide holder into the tray
  2. 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 °C.
  3. 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.
  4. 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.
  5. Repeat the wash step with fresh 1x wash buffer for 2 min at RT.

7. Hybridize RNA-ISH amplifier 3

  1. Remove excess liquid from the slides. Remove the humidity control tray from the oven and place the slide holder into the tray
  2. 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 °C.
  3. 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
  4. 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.
  5. Repeat the wash step with fresh 1x Wash Buffer for 2 min at RT.

8. Hybridize metal oligos

  1. 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).
  2. Close the tray and insert it into the hybridization oven for 45 min at 40 °C.
  3. Remove the humidity control tray from the hybridization oven and remove the slide holder. Return the tray to the oven.
  4. 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.
  5. Repeat the wash step with fresh 1x Wash Buffer for 2 min at RT.

9. Metal conjugated antibodies

  1. To block non-specific sites, apply enough volume of blocking buffer to cover the entire tissue section and incubate for 30 min at RT.
  2. During incubation, prepare the antibody mix to be used in step 10.1 by diluting the antibodies (Table 1) in Antibody diluent buffer.

10. Incubation with metal conjugated antibodies

  1. 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.
  2. Place the slide in a humidity chamber and incubate overnight (~16 h) at 4 °C.

11. DNA staining

  1. Prepare fresh Ir-Intercalator solution.
    1. From a 500 µM stock solution, prepare a 1:2000 working solution in RNase-free TBS. Working solution can be aliquoted and stored at -20 °C (do not freeze-thaw).
  2. 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.
  3. Repeat wash in TBS-T.
  4. Stain slides with Ir working solution (from step 11.1.1) for 5 min at RT.
  5. 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.
  6. Repeat wash in TBS-T.
  7. Wash twice in TBS, 5 min (stir slowly).
  8. Dip the slide quickly in ddH2O to avoid crystallization of the salts present in the TBS on the tissue.
  9. Dry the slide under a chemical hood (10 min) and store at 4 °C until image acquisition.

12. Image acquisition

  1. Load the slide into the ablation chamber of IMC.
  2. Capture a panorama image to identify the region of interest (ROI).
  3. Map out ROIs in the image acquisition software.
  4. Apply the acquisition template.
  5. 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 µm x 30 µm), 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.
  6. 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.
  7. Measure the quantity of each isotope that is measured by the detector and converted into data.

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Results

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 ...

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Discussion

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...

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Disclosures

Jared K. Burks is a consultant for Standard BioTools.

Acknowledgements

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's Cancer Center Support Grant P30 CA016672. This research was also supported in part by the MD Anderson's Cancer Center'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.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
 https://www.protocols.io/view/oligonucleotide-polymer-conjugation-for-imaging-ma-dm6gpjz91gzp/v2.Protocols.IOdx.doi.org/10.17504/protocols.io.dm6gpjz91gzp/v2Oligonucleotide-polymer conjugation for imaging mass cytometry V.2 
10x TBS RNAse-FreeThermo Fisher Scientific J62780.K7For intermittent washes between steps
ACD HybEZ II Hybridization SystemACD Biotechne321711Oven for baking tissue slides
Antibody DiluentDakoS0809 Antibody diluent buffer
Antibody: B7-H3BethylA700-025CF, A700-026CFClone: BLR025F, BLR026F
Antibody: B7-H4Thermo Fisher Scientific 14-5949-82Clone: H74
Antibody: BRD4(6F11)BethylA700-005CFClone: BL-151-6F11
Antibody: CCR6R&DMAB195-100Clone: 53103
Antibody: CD11bAbcam ab209970Clone: EPR1344
Antibody: CD25Abcam ab215378Clone: EPR6452
Antibody: CD278/ICOSCST 89601BFClone: D1K2T
Antibody: CD31Abcam ab207090Clone: EPR3094
Antibody: CD33Abcam ab238784Clone: SP266
Antibody: CD3e CST85061BFClone: D7A6E
Antibody: CD4Abcamab181724Clone: EPR6855
Antibody: CD44Thermo Fisher Scientific MA1-10225Clone: IM7
Antibody: CD45Thermo Fisher Scientific 14-9457-82Clone: CD45-2B11
Antibody: CD8aCST 85336BFClone: D8A8Y
Antibody: CXCL13R&DAF801Clone: Polyclonal
Antibody: CXCR5R&DMAB190-100Clone: CXCR5
Antibody: gamma-H2AXBethylA700-053CFClone: BLR053F
Antibody: Granzyme BSanta Cruzsc-8022 xClone: 2C5
Antibody: KAP1BethylA700-014CFClone: BL-248-2G6
Antibody: Ki67BethylA700-021CFClone: BLR021E
Antibody: LCK CST2984BFClone: D88
Antibody: MFAP5N/AN/APMID: 31332047 / Clone: 130A  (Note: custom designed) 
Antibody: SMACST56856BFClone: 1A4
Antibody: VEGFSTB-Fluidigm3163028DClone: G153-494
Antibody: VISTACST64953BFClone: D1L2G
AZ-AR1 Elegance Citra BioGeneXHK546-XAKAntigen retrieval buffer
Ethyl Alcohol 100% (200 Proof)Pharmco111000200For deparaffinization
EZ – Retriever IRBioGeneXMW016-IRAntigen Retrieval System for FFPE Tissue Sections
HybEZ II Hybridization SystemACD Biotechne321710The 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)
ImmEdge Hydrophobic Barrier PAP Pen VectorLabsH-4000Hydrophobic Barrier Pen
Ir Intercalator SolutionThermo Fisher Scientific NC1038184Used for identifying nucleated cells
KERATIN 8/18CST 4546BFClone: C51
Metal OligosVariousVariousMetal tag ranging from T1 to T12 depending on which is needed
Nuclease-free water (NFW)Corning 46-000-CM To clean and prep all equipment, and to use as a dilutant when necessary
RNAscope Protease Plus ACD Biotechne322331 Used to permeabilize cell membranes of samples
RNAscope EZ-Batch Slide HolderACD Biotechne310017Designed to manually batch process up to 20 slides efficiently
RNAscope HiPlex12 Reagents Kit v2 ACD Biotechne324409Reagents for RNAScope
RNAscope HiPlex Probe - Target ProbesACD BiotechneVariousRange from T1 to T12 dependent on which is needed
RNaseZap RNase Decontamination SolutionThermo Fisher ScientificAM9782A surface decontamination solution that destroys RNases
Staining Rack Microscope Slide Basket Holder 24 Place Chemical Resistant PlasticOptimal Scientific3028For staining multiple slides
Tween 20Thermo Fisher Scientific AAJ20605APFor intermittent washes between steps
Ultravision Protein BlockThermo Fisher Scientific TA125PBQBlocking buffer

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Spatial TranscriptomicsIn Situ HybridizationMetal Probe DetectionOvarian Cancer BiomarkersCD8 T CellsCancer Associated FibroblastsSpatial Proteomics
Dual-modality Molecular Cartography: Integrating Multiplex mRNA Detection with Protein Imaging Mass Cytometry
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