In Situ Hybridization (ISH) Services: RNA ISH & FISH

Introduction

In situ hybridization (ISH) lets you detect and localize specific RNA or DNA targets directly in tissue. Because it preserves tissue structure, it shows where a gene is expressed—not just whether it is present. This helps map gene expression to specific cell types within a tissue section.

In this guide, you’ll learn how ISH works, when to use RNA ISH vs DNA ISH, and how CISH and FISH compare.

If you want to outsource ISH, we can run the full workflow—from sample guidance (FFPE or frozen) and assay setup to imaging, QC, and report-ready results. To start, send your target, species, sample type, and sample count. We’ll confirm what’s possible, share turnaround time, and provide a quote.

What Is In Situ Hybridization (ISH)? How It Works

Figure 1. Example result from our in situ hybridization (ISH) service: RNA ISH detection of PP1B mRNA with clear signal and preserved morphology. Click the image to view the full-resolution slide in our Whole Slide Imaging (WSI) portal.

In situ hybridization (ISH) uses labeled probes to detect specific RNA or DNA in tissue. It works on FFPE and frozen sections. Because the tissue stays intact, ISH shows where a target appears and which cells generate the signal. Bulk methods like qPCR or RNA-seq can’t show this spatial context. By detecting nucleic acids within each tissue section, ISH helps you interpret results in place.

In cancer samples, ISH can show whether a localized gene expression signal comes from tumor cells or another specific cell type. It can also reveal how expression levels vary across different regions within the same tissue section.

How Does In Situ Hybridization (ISH) Work? Step-by-Step

To get a clean signal and consistent results, we follow a standard ISH workflow and fine-tune key settings for each tissue type and target.

1. Sample Preparation & Pre-treatment: We prepare tissue sections (FFPE or frozen) and run target retrieval and permeabilization. This keeps morphology intact while helping probes reach nucleic acids.

2. Probe Hybridization: We apply specific RNA or DNA probes under controlled temperature and buffer conditions so they bind the correct target with high specificity. When needed, we use probes designed for your sequence, including RNA targets.

3. Stringency Washes & Signal Development: We use stringency washes to reduce background. When needed, we add signal amplification to improve sensitivity and contrast.

4. Detection & Imaging (CISH/FISH): We visualize targets using chromogenic ISH (CISH) or fluorescent ISH (FISH) detection. We then image the slides (including whole slide imaging, WSI) and perform QC review to confirm results are easy to interpret.

In our in situ hybridization (ISH) services, we tailor pre-treatment, probe conditions, and detection chemistry to your sample and study goal. You’ll receive report-ready images with clear QC notes and tissue-contextual interpretation.

Automated Staining for Consistency (Leica BOND)

Automation improves consistency and throughput. We use systems such as Leica BOND (when appropriate) to standardize timing, temperature, reagent handling, and washes. This reduces variability and supports stable signal quality across batches—especially in large or multi-timepoint studies.

Deliverables (What You’ll Receive) & Quality Control

We provide clear, report-ready results that are easy to review and share. Depending on your study, what you receive may include:

• Report-ready images (selected fields of view and/or representative regions)

• Optional Whole Slide Imaging (WSI) access for remote review and collaboration

• A short QC summary describing slide quality, signal-to-background, and key technical notes

• Optional quantification (when needed), such as spot counting or region-based metrics for RNA ISH, and signal enumeration/scoring for FISH.

QC checkpoints (examples)

QC checkpoints include morphology/background review, control verification, and consistency checks across batches or cohorts (when applicable). We align QC criteria and deliverables upfront and provide high-resolution images, optional WSI access, and analysis tables/reports based on your workflow.

Common Applications of ISH (RNA ISH and FISH)

Common ISH applications include:

• Spatial validation of RNA-seq targets in FFPE tissue

• Cell-type localization in heterogeneous tumors

• Viral RNA/DNA detection in tissue

• Biomarker development and IND-enabling studies

• Copy number changes (amplification) via FISH

• Structural variants (translocations) via FISH

Choosing the Right Assay: ISH vs IHC (and when to use FISH)

Not sure whether to use IHC or ISH? IHC detects proteins, while ISH detects RNA/DNA in tissue. The table below summarizes key differences. It shows when fluorescence in situ hybridization (FISH)—a DNA ISH method—is best for copy number changes and translocations.

Feature	RNA ISH (e.g., CISH/fluorescent RNA ISH)	IHC	DNA ISH (FISH)
Detects	mRNA / lncRNA	Proteins	DNA sequences
Best for	Spatial gene expression, cell-type localization	Protein abundance & localization	Copy number, translocations, amplifications
Typical readout	Dots/signal in cells; tissue context preserved	Staining intensity/pattern	Discrete fluorescent signals; genomic loci
Sample types	FFPE, frozen (depends on assay)	FFPE, frozen	FFPE, fresh/frozen (depends)
Strengths	Adds spatial context to transcript data	Cost-effective, widely available	Strong for genomic alterations
Limitations	Sensitive to RNA quality & pre-analytics	Antibody specificity/epitope masking	Requires fluorescence imaging; interpretation expertise

RNA ISH is a strong choice when you need to confirm gene expression in context. It also helps link expression to cell types within a tissue section.

Quick decision guide

• Choose IHC when you need protein expression in tissue.

• Choose RNA ISH when you need to see where transcripts are expressed and which cells produce the signal.

• Choose FISH (DNA ISH) when you need copy-number changes or structural variants (e.g., amplification or translocation).

Not sure which assay fits your target and sample type? Send us your target and sample details. We’ll recommend the best option and confirm sample needs before you ship tissue.

Sample Requirements for ISH (FFPE & Frozen)

To get a reliable ISH signal and clear tissue context, share your sample type, species, and target details before you ship. We accept FFPE blocks or unstained slides. We may also support frozen tissue/sections, depending on the project. When possible, avoid heavily baked slides or long room-temperature storage. These conditions can reduce RNA quality and weaken signal.

Recommended submission formats

• FFPE: Block or unstained slides (typical section thickness 3–7 µm)

• Frozen: Fresh-frozen tissue or cryosections (requirements vary by assay)

Information to include when requesting a quote

• Target (gene name/ID; transcript/region if known) and whether you need RNA ISH or DNA ISH (FISH)

• Species and tissue type (organ, disease model)

• Number of samples/slides and study design (control vs treated, time points)

• Preferred readout (chromogenic vs fluorescent) and whether quantification is needed

• Probe design needs: target ID/sequence region (if known), species, and any preferred probe set/vendor

If you’re unsure what to send, share your study goal and sample details. We’ll recommend the best format and confirm requirements before you ship.

Why Outsource to a Specialized In Situ Hybridization Service?

ISH is sensitive to sample handling and assay conditions, especially in FFPE tissue. A specialized ISH service reduces variability and helps you avoid reruns. It also delivers clear results faster, so you can focus more on interpretation and less on troubleshooting.

Get Started with iHisto ISH Services

To get started, send us your target(s), species, tissue type, sample format (FFPE block/slides or frozen), and sample count. We’ll confirm what’s possible, recommend controls, share turnaround time, and send a quote.

GLP-Compliant Excellence for IND-Enabling Studies

For IND-enabling work, teams need strong documentation and traceability. iHisto offers GLP-aligned histology and ISH services with QA oversight (when needed) to support regulated study workflows.

Scaling with Automated Staining and Quantitative Image Analysis

We combine staining with quantitative image analysis. Using standard workflows and tools like HALO, when needed, we turn images into reproducible metrics. These metrics support comparisons across groups and time points.

Frequently Asked Questions (FAQ)

• What is the turnaround time for in situ hybridization (ISH) services?

  Most projects finish in ~2–3 weeks. Timing depends on sample type (FFPE vs frozen), target abundance, probe availability (provided vs custom), and any assay optimization. If you have a fixed deadline, rush options may be available.

• Do you support FFPE samples for RNA ISH or DNA ISH?

  Yes. We routinely run RNA ISH and DNA ISH on FFPE tissue sections. Typical section thickness is 3–7 µm. We can also advise on fixation, block age, and slide preparation for archival samples.

• What sample types do you accept for ISH?

  We commonly accept FFPE blocks or slides. We may also support frozen tissue/sections, depending on the project. If relevant, we can discuss cell pellets/cell blocks as well.

• Do you offer RNA ISH, DNA ISH, CISH, and FISH?

  Yes. We offer RNA ISH and DNA ISH with both chromogenic ISH (CISH) and fluorescence in situ hybridization (FISH) options. We’ll recommend the best format based on your target (expression vs genomic alteration), tissue type, and multiplexing needs.

• Do you design probes, or can I provide my own probes?

  Either option works. You can provide validated probes. Or we can discuss probe sourcing and design based on species, target sequence, and assay needs. We’ll confirm target details and recommended controls before starting.

• What controls are recommended for ISH experiments?

  Controls depend on your target and sample type. Common controls include:

  - Positive control (known-expressing tissue/cell line)

  - Negative control (sense probe or background control, as appropriate)

  - Housekeeping/quality control target to assess RNA integrity (for RNA ISH). We can recommend a control plan for your study.

• What deliverables will I receive?

  You’ll receive report-ready images and a short QC/notes summary. If requested, we can provide WSI access for review and collaboration. Annotated fields of view can be included when needed.

  
  

• Can you quantify ISH results?

  Yes, when quantification fits the signal pattern. Options include spot counting for RNA ISH (per cell or per area). For FISH, we can provide signal enumeration/scoring, such as copy-number metrics. We can also provide region-based analysis and standardized reporting.

• How is pricing determined for ISH services?

  Pricing depends on the sample count and tissue type. It also depends on the assay format, like chromogenic or fluorescent. Pricing can change based on probe needs and multiplexing. Imaging or whole-slide imaging may affect the price. The price also depends on whether quantification is included. Share your study outline and we’ll provide a quote.

• Can ISH be performed under GLP-aligned workflows for IND-enabling studies?

  Yes, depending on study design and documentation needs. We can support GLP-aligned practices for IND-enabling programs, including traceability and QA oversight (as required). Tell us your requirements upfront so we can align deliverables.

  
  

• Do you support multiplex RNA ISH or multi-target assays?

  Yes. Multiplex options depend on tissue type, target expression, and detection format (chromogenic vs fluorescent). Share your target list and desired number of channels, and we’ll recommend an assay design and controls.