RNA - protein co-detection with Imaging Mass Cytometry and RNAscope assay

Figure 1: Pan-keratin CD68 CXCL10 CXCL13 Breast cancer

Figure 1: Pan-keratin, CD68, CXCL10, CXCL13 - Breast cancer

Figure 2: α-SMA Ki67 CXCL9 VEGFA Breast cancer

Figure 2: α-SMA, Ki67, CXCL9, VEGFA - Breast cancer

Figure 3: α-SMA CD45 CD68 CXCL13 VEGFA Breast cancer

Figure 3: α-SMA, CD45, CD68, CXCL13, VEGFA - Breast cancer

Figure 4: α-SMA CD45 CD68 GAPDH ACTB Tissue-Breast cancer

Figure 4: α-SMA, CD45, CD68, GAPDH, ACTB - Breast cancer

Tumor-immune interaction in breast cancer tissue using RNAscope HiPlex with Imaging Mass Cytometry for RNA-Protein co-detection. RNAscope was used for visualizing chemokines and growth factors while antibodies were used for immune cell markers.


RNAscope™ HiPlex assay can be combined with Imaging Mass Cytometry™ (IMC™) to enable same-slide co-detection of RNA and protein targets on FFPE tissues.

IMC is a proven tool for the study of complex cellular interactions in the TME. It utilizes CyTOF® technology for simultaneous assessment of 40-plus protein markers at subcellular resolution without spectral overlap or background autofluorescence, thus providing unprecedented insight into the tissue organization and function. The RNAscope HiPlex v2 assay can detect up to 12 targets on FFPE tissues. By using metal-conjugated labeled probes instead of the Fluoros ( Fluoros T1-T4, Fluoro T5-T7, Fluori T8-T12), RNA targets can be detected using the Hyperion Imaging Mass Cytometry system.

Graphical schematic of the three-step procedure to acquire RNA and protein co-detection data with IMC




Key advantages of RNA-protein co-detection

  • Spatial phenotyping with cell marker-specific antibodies and target RNA expression.
  • Visualizing inflammatory signatures.
  • Studying cell-type specific gene expression.
  • Identifying the cellular source of secreted factors such as cytokines, chemokines and growth factors.

This technical note provides a workflow and procedure to achieve RNA and protein co-detection data in IMC experiments. This workflow utilizes the highly sensitive and specific RNAscope technology for RNA detection with the multiplexing capability of IMC to visualize key RNA and protein markers in the same FFPE samples



RNA - protein co-detection with Imaging Mass Cytometry using the RNAscope assay

Tech Note

Co-Detection of RNA and Protein to Explore Tumor-Immune Interactions Utilizing RNAscope With Imaging Mass Cytometry


To learn more about the RNAscope™ HiPlex12 v2 Assay - please click here: https://acdbio.com/rnascope-hiplex-assays or review the user manual here


Getting started:

Step1: Here are the reagents needed for Step 1 of the workflow


Metal-ready probes (ACD, Bio-Techne)

Product Information

RNAscope™ HiPlex metal-ready probes T1-T12


RNAscope™ HiPlex metal-ready probe T1


RNAscope™ HiPlex metal-ready probe T2


RNAscope™ HiPlex metal-ready probe T3


RNAscope™ HiPlex metal-ready probe T4


RNAscope™ HiPlex metal-ready probe T5


RNAscope™ HiPlex metal-ready probe T6


RNAscope™ HiPlex metal-ready probe T7


RNAscope™ HiPlex metal-ready probe T8


RNAscope™ HiPlex metal-ready probe T9


RNAscope™ HiPlex metal-ready probe T10


RNAscope™ HiPlex metal-ready probe T11


RNAscope™ HiPlex metal-ready probe T12



Standard BioTools:

Labelling kit (Standard BioTools)

Product Information

Maxpar X8 Antibody labeling kit

Select the metal-specific kit of your choice


Step2: Here are the reagents needed for Step 1 of the workflow


Assay (ACD, Bio-Techne)

Product Information

RNAscope™ HiPlex12 Flex Reagents Kit v2 (without Fluoros) 


RNAscope HiPlex12 Reagents Kit v2 (with Fluoros)







Equipment (ACD, Bio-Techne)

Product Information

HybEZ™ II Hybridization System

321710 or 321720

HybEZ Humidity Control Tray (with lid)



Standard BioTools

Labelling kit (Standard BioTools)

Product Information

Maxpar X8 Antibody labeling kit

Select the metal-specific kit of your choice


Step3: Please visit the Standard BioTools website to get the suitable antibody panels.

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