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Related Links
Dual RNAscope ISH-IHC Antibodies
Primary Antibodies for IHC
Secondary Antibodies - HRP Polymer
RNAscope ISH Technology
Chromogenic vs Fluorescent Detection
HRP-Polymer Detection Tech Note
Dual ISH-IHC Webinar
IHC Handbook
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Dual RNAscope® ISH-IHC (in situ hybridization – immunohistochemistry) allows scientists to simultaneously detect mRNA and protein expression in tissues. This multi-omics approach broadens our understanding of the regulation, production, and localization of RNA and proteins in specific tissue types. By coupling RNAscope® ISH with antibody-dependent IHC, a more complete analysis of the molecular mechanisms involved in biological pathways can be undertaken to reveal key cell-cell interactions. Bio-Techne’s RNAscope® ISH-IHC Co-Detection Assays virtually eliminates the costly and time-consuming optimization often required for detection of RNA and proteins on single tissue sections, preserving precious samples.
View Our Dual ISH-IHC Validated Antibodies
Benefits of Dual RNAscope™ ISH-IHC
- Validate Antibody Specificity. Comparing localization of protein and its mRNA in the same tissue qualifies as orthogonal antibody validation.
- Visualize Source of Secreted Proteins. Target the transcript of secreted proteins, such as cytokines or chemokines, and use protein markers to identify the cellular source.
- Ascertain Marker Expression, Activation, and Spatial Mapping. Detection in a morphological relevant context is used to assess cell-cell interactions (i.e. hormone-receptor interaction, endocytosis or excretion). Immune cells that infiltrate the tissue microenvironment (TME) can be characterized using activation markers detected by ISH and immune cell markers such as CD3, CD4, CD8, CD68, and CD45 by IHC. Similarly, this dual approach can be applied to brain mapping to detect specific gene expression in neuronal subtypes (ISH) alongside standard neuronal markers (IHC).
- Increase Multiplexing Capability. Several markers are visualized simultaneously in tissue and ISH is employed when an adequate antibody is not available.
- Identify Transcript Variants. Detect cell type specific expression of splice variants and gene mutations.
Enhanced ISH-IHC Co-Detection Assays Broaden Multiplexing Capabilities
ISH and IHC are complementary techniques, bridging the gap between RNA and protein analysis. Yet, protocol optimization for dual mRNA and protein detection may be quite challenging, requiring antibody screening or the use of sequential slides when a preferred antibody clone is incompatible with ISH conditions. With the improved co-detection workflow, successfully combining an IHC-validated antibody with ISH on a single tissue section is vastly increased. Additionally, antibody specificity is retained with this new protocol, producing staining results equivalent to those observed from IHC alone.
Considerations for Tissue Preparation and Antigen Retrieval
- Freshly-cut tissue sections are required for ISH ( < 2 weeks since sectioned and mounted onto histological slides).
- Establish a working IHC protocol before proceeding to ISH. The primary antibody concentration may need to be adjusted within the context of ISH.
- Animal tissue can be used for both chromogenic and fluorescent IHC. However, many human tissues have endogenous autofluorescence and thus require optimization when using fluorescently-conjugated antibodies in human tissue.
- Fresh or fixed frozen tissue have less autofluorescence compared to formalin-fixed paraffin embedded (FFPE) prepared tissues. While either chromogenic or fluorescent detection can be used with frozen tissues, chromogenic detection is preferred for FFPE tissues due to high background fluorescence.
- FFPE samples are more tolerant to heat-induced epitope retrieval treatments than frozen samples. Frozen samples are susceptible to physical degradation at high temperatures and thus require optimization using a lower temperature for retrieval.
- Control tissue slides from Novus and probes from ACD provide users with positive and negative controls.
ISH and IHC Basics
In situ hybridization (ISH) technology amplifies target-specific probes to visualize RNA expression. Immunohistochemistry (IHC) can use chromogenic detection or fluorescent detection methods to determine expression and localization of proteins in FFPE or frozen tissue, depending on target abundance and accessibility.
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