In Situ Hybridization: High-Sensitivity RNA and DNA Detection in Tissue

Overview

In situ hybridization (ISH) is a powerful molecular technique used to detect specific RNA or DNA sequences directly within tissue sections. By using labeled nucleic acid probes, ISH enables the visualization of gene expression or pathogen presence at the cellular level while preserving spatial architecture.

At iHisto, we specialize in RNA- and DNA-based ISH on FFPE and frozen samples, combining expert probe design, clean signal development, and Leica BOND RX automation for consistency and reproducibility in every slide.

How In Situ Hybridization Works

ISH involves hybridizing a labeled complementary probe to a target RNA or DNA sequence in tissue. Once bound, the probe is detected using chromogenic or fluorescent methods, allowing visualization of the transcript or genetic element in context.

Probe Types Supported:

• RNA probes (e.g., mRNA, lncRNA, viral RNA)

• DNA probes (e.g., gene fusions, repeats, CNVs)

• Custom antisense oligonucleotide or locked nucleic acid (LNA) probes

Detection Modes:

• Chromogenic (CISH) – enzyme-based colorimetric readout

• Fluorescent (FISH/IFISH) – multiplexed or high-resolution detection

• TSA-Amplified ISH – for ultra-low expression targets

Step-by-Step Workflow

1. Sample Preparation
   Sections from FFPE or frozen tissue are cut, mounted, and processed using our Leica BOND RX system for fully automated hybridization and detection.

2. Pretreatment & Digestion
   Tissues are prepped to remove barriers and improve probe access.

3. Probe Hybridization
   Labeled probes are applied to target specific nucleic acid sequences.

4. Signal Detection
   Chromogenic or fluorescent reporters are used to detect hybridized probes.

5. Counterstaining
   Hematoxylin or DAPI provides tissue or nuclear contrast.

6. Mounting & Imaging
   Slides are coverslipped and scanned for analysis.

iHisto’s Optimized ISH Services

• ✅ RNA or DNA probe targeting

• ✅ Compatible with FFPE and frozen samples

• ✅ Chromogenic or fluorescent signal development

• ✅ Leica BOND RX automation for high-throughput and reproducibility

• ✅ Pathologist-reviewed results and digital image delivery

Applications of In Situ Hybridization

ISH is widely used in both clinical and research settings to localize and quantify gene expression or pathogen markers.

Common use cases:

• Gene expression profiling (e.g., cytokines, oncogenes)

• Detection of viral RNA (e.g., CMV, HPV, SARS-CoV-2)

• Microbial localization (bacterial 16S/18S rRNA)

• Chromosomal translocations or fusions

• Cell-type marker analysis via RNA transcripts

ISH Formats and Kits Supported at iHisto

We support multiple in situ hybridization workflows depending on your application and target expression level:

• ✅ CISH (HCR kit) – Chromogenic in situ hybridization using hybridization chain reaction (HCR) for signal amplification

• ✅ CISH (ACD kit) – Chromogenic detection using RNAscope technology from ACD for RNA visualization in FFPE tissue

• ✅ FISH (ACD kit) – Fluorescent in situ hybridization using multiplex or single-target RNA detection with fluorescence output

These kits can be integrated with Leica BOND RX automation for consistent, high-quality results.

FAQs

• What’s the difference between IHC and ISH?
  IHC detects proteins, while ISH detects nucleic acids (RNA/DNA). ISH is ideal when gene expression data is needed or when no good antibody is available.

• Do you offer custom probe design?
  Yes. We can assist with custom probe development and validation, or work with probes you provide.

• Can ISH be combined with IHC?
  Yes, we can perform dual ISH-IHC or ISH-IF protocols to localize gene expression alongside protein markers.

• What samples do you accept?
  We support FFPE and frozen tissue from human, rodent, NHP, and xenograft studies.

Ready to Detect RNA or DNA in Tissue?

At iHisto, our in situ hybridization services bring precision and spatial context to molecular detection. Whether you're studying gene expression, infections, or rare transcripts, we provide clear results that advance your research.

📩 Contact us to request a quote or consultation.