Home › Cell Biology › Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography
Cell Biology JoVE (Open Access) Citable · DOI

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

DOI: 10.3791/58293-v
What you'll learn
  • ✓ Prepare millimeter-scale specimens for micro-CT imaging via fixation and dehydration
  • ✓ Embed stained tissue samples in acrylic resin using custom apparatus
  • ✓ Achieve rigid immobilization and long-term storage for 3D histological analysis
Protocol

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

Difficulty
intermediate
Total time
~3–4 days per specimen (includes fixation, dehydration, staining, infiltration, and embedding)
Model organism
Fish (zebrafish or similar teleost)
Biosafety
BSL-1

Steps

1
Fix fish specimens using appropriate fixative

Prepare whole fish specimens by immersion in fixative solution to preserve tissue architecture and cellular morphology. This step stabilizes proteins and prevents autolysis.

▶ 01:05
2
Dehydrate and stain fixed specimens

Remove residual water via graded ethanol series and apply histological stains to enhance tissue contrast for micro-CT visualization. Staining improves radiodensity differentiation between tissue types.

▶ 01:48
3
Infiltrate specimens with resin monomer

Gradually introduce acrylic resin monomer into dehydrated tissue to displace organic solvents and prepare specimens for polymerization. This ensures uniform embedding medium penetration.

▶ 02:24
4
Embed specimens in rigid acrylic resin

Transfer infiltrated specimens into polyimide tubing using the custom-designed embedding apparatus and polymerize resin to achieve rigid, stable immobilization suitable for long-term storage and repeated micro-CT scanning.

▶ 03:07
5
Validate embedding quality via micro-CT imaging

Scan embedded specimens using micro-CT to confirm successful embedding, absence of voids or artifacts, and preservation of tissue morphology for subsequent 3D histological analysis.

▶ 05:18
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