4.2.1 Frozen sectioning

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4.2.1 Frozen sectioning

Keiji Inohaya
Tokyo Institute of Technology

4.2.1.1 Equipment and reagents

• Eecrtro Feeze MCR802A (Komatsu Electronics)
• Microtome ROM-380 (Yamato)

• 4% paraformaldehyde in phosphate-buffered saline (PBS)
• PBST (0.1% Tween-20 in PBS)
• 15% sucrose in PBS
• 7.5% gelatin / 15% sucrose in PBS

• APS-coated slide glass

4.2.1.2 Method

1. Fix specimens with 4% paraformaldehyde in phosphate-buffered saline (PBS) overnight at 4 ºC.
2. Wash 3 times in PBST (0.1% Tween-20 in PBS).
3. Transfer specimens into 15% sucrose in PBS overnight at 4 ºC.
4. Incubate in 7.5% gelatin / 15% sucrose in PBS at 37 ºC.
5. Embed specimens in 7.5% gelatin / 15% sucrose in PBS at 4 ºC. The embedded specimens can store for weeks at 4 ºC.
6. Orientate gelatin blocks with a knife.
7. Freeze blocks, and section at 10-20 µm using a microtome
8. Transfer sections on to a drop of water on APS-coated slide.

4.2.1.3 Reference

Inohaya K, Yasumasu S, Ishimaru M, Ohyama A, Iuchi I, and Yamagami K (1995). Temporal and Spatial Patterns of Gene Expression for the Hatching Enzyme in the teleost Embryo, Oryzias latipes. Dev. Biol. 171, 374-385.