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Analytical Chem JoVE (Open Access) Citable · DOI

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

DOI: 10.3791/2533-v
What you'll learn
  • ✓ Perform whole mount in situ hybridization (WISH) on zebrafish embryos
  • ✓ Visualize spatial gene expression patterns using riboprobe detection
  • ✓ Integrate molecular biology techniques with organismal comparative anatomy
  • ✓ Interpret developmental gene expression in vertebrate model organisms
Protocol

Whole mount in situ hybridization (WISH) was used in an upper level undergraduate Comparative Vertebrate Biology course in addition to vertebrate dissections. This gave students the opportunity to study gene expression patterns as well as gross anatomy, linking the study of molecular and organismal biology within one course.

Difficulty
intermediate
Total time
~3–4 days (including fixation, hybridization overnight, and antibody incubation steps)
Model organism
Zebrafish (Danio rerio)
Biosafety
BSL-1

Steps

1
Fix embryos and digest with proteinase K

Fixed zebrafish embryos are treated with proteinase K to permeabilize tissue and allow riboprobe penetration. Proper digestion time is critical to balance access while preserving morphology.

▶ 02:04
2
Hybridize riboprobes to target sequences

Digoxigenin-labeled riboprobes are incubated with embryos under stringent conditions to bind complementary mRNA sequences. Overnight hybridization at controlled temperature ensures specific gene expression detection.

▶ 05:34
3
Block and incubate anti-digoxigenin antibodies

Non-specific binding sites are blocked, then anti-DIG alkaline phosphatase–conjugated antibodies are applied to recognize hybridized riboprobes for visualization.

▶ 07:42
4
Stain and process final specimens

Alkaline phosphatase substrate is added to generate colored precipitate at sites of probe binding. Specimens are then cleared and mounted for microscopic imaging.

▶ 09:11
5
Analyze gene expression patterns in zebrafish

Resulting whole mount preparations reveal spatial distribution of aldh1a2, fgf8a, and pax2a expression, linking molecular signals to embryonic developmental anatomy.

▶ 10:58
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