Optimizing your in situ hybridization for success

August 30, 2024

Sofie Van Rossom, PhD

What is In Situ Hybridization (ISH)?

in situ hybridization (ISH) is a technique that enables the localization of specific DNA or RNA sequences within tissue sections and cytology samples using labeled complementary strands. The target gene can be visualized through brightfield or fluorescence microscopy. Compared to immunohistochemistry (IHC), which broadly localizes proteins within tissues, ISH offers greater sensitivity, allowing researchers to understand the organization and function of genes in tissue sections. ISH not only identifies cells positive for a specific DNA/RNA sequence but also quantifies the level of positivity within individual cells.

How can you benefit from combining ISH and IHC assays

ISH is a powerful tool in both preclinical research and clinical trials, offering critical insights. It can be combined with IHC assays to provide unique perspectives on cellular and molecular mechanisms within tissues.

Key applications include:

• Relationship Between Targets: Dual assays on unique but related targets reveal their interrelationships.
• Protein Localization: Identify the location of a target protein within tissue and the originating cells.
• Gene Regulation Insight: Combining ISH and IHC on the same target provides insights into gene regulation mechanisms.
• Tumor Region Analysis: Use IHC cytokeratin markers with ISH assays to highlight gene regulation in tumor regions.
• Protein Degradation Insights: Dual assays offer valuable insights into protein degradation processes.

Maximizing your in situ hybridization assays

Successful ISH assays depend on meticulous sample preparation to minimize RNA degradation in tissue.

• Tissue Fixation: Fix tissues promptly in 10% Neutral Buffered Formalin (NBF) at 20x the tissue sample volume, then transfer to 70% ethanol (EtOH).
• RNA/DNA Preservation: Cut fresh sections under RNase/DNase-free conditions onto charged slides for better adhesion, and store at a constant 4°C or at room temperature.
•  Baking conditions: Bake slides for 30-120 min at 60°C for better adhesion.

Another crucial aspect for a successful ISH assay is optimization. Each unique combination of species, tissue type, and gene target must be optimized to hone in on the ideal conditions for detecting and visualizing the target. Pre-existing IHC data can guide the optimization process.

Additionally, digital whole slide imaging settings must be tailored to each sample set, accounting for tissue type differences, stain intensity, and autofluorescence.

Consistency in applying the ISH protocol across samples is key. Automated technologies, like the Leica BondRX and Ventana Discovery Ultra, ensure precise and consistent application, minimizing human error.

Leveraging AI-Powered Data for your ISH Assays

The integration of AI-powered algorithms with digital whole slide ISH images enhances the analysis, generating data and insights beyond human capability. However, several key steps ensure successful ISH assays.

Our AI-powered algorithms ensure optimal nuclear segmentation for the most reliable results.

Rest assured that our in-house team of pathologists work in proximity with our imaging specialists to ensure you get optimized data from your ISH assays, that signal is neither undercounted nor overestimated.

By leveraging cutting-edge ISH technologies, CellCarta ensures comprehensive and high-quality results for your research and diagnostic needs.

Contact us to get your project started.

 

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