Cell Biology JoVE (Open Access) Citable · DOI

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

Name: Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue
Uploaded: 2026-06-23
Duration: 17 min 38 s
Description: Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. ...

DOI: 10.3791/3764-v

What you'll learn

✓ Perform radioactive in situ hybridization to detect mRNA spatial patterns in tissue
✓ Prepare tissue sections and apply pre-hybridization treatments for optimal probe binding
✓ Generate darkfield color images to visualize and quantify gene expression patterns

Protocol

This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.

Difficulty

advanced

Total time

~5–7 days (including probe synthesis, tissue preparation, hybridization, and signal detection)

Model organism

Avian embryo (chick)

Biosafety

BSL-1

Steps

Prepare tissue sections for in situ hybridization

Collect and section embryonic tissue on slides. This foundational step ensures tissue integrity and accessibility for probe hybridization.

▶ 02:16

Apply pre-hybridization treatment to tissue

Treat tissue sections with proteinase K and other reagents to permeabilize membranes and reduce background signal. This enhances probe penetration and specificity.

▶ 03:20

Hybridize radioactive probe to target mRNA

Incubate labeled probe with tissue sections under controlled temperature and humidity. The probe binds to complementary mRNA sequences in the tissue.

▶ 04:36

Visualize radioactive signal via autoradiography

Expose emulsion-coated slides to detect radioactive decay and develop the signal. This produces a visible map of mRNA localization in tissue.

▶ 07:34

Generate darkfield color images of expression

Acquire and process darkfield microscopy images to create high-contrast visualizations of mRNA distribution. This enables quantitative analysis of gene expression patterns.

▶ 10:59

Interpret mRNA expression patterns in tissue

Analyze and document spatial gene expression results in the experimental tissue (avian brain in this example). Results demonstrate method utility for detecting low-abundance transcripts across multiple tissues.

▶ 11:38

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