4.2.1 Frozen sectioning
Tokyo Institute of Technology
188.8.131.52 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
- Fix specimens with 4% paraformaldehyde in phosphate-buffered saline (PBS) overnight at 4 ºC.
- Wash 3 times in PBST (0.1% Tween-20 in PBS).
- Transfer specimens into 15% sucrose in PBS overnight at 4 ºC.
- Incubate in 7.5% gelatin / 15% sucrose in PBS at 37 ºC.
- Embed specimens in 7.5% gelatin / 15% sucrose in PBS at 4 ºC. The embedded specimens can store for weeks at 4 ºC.
- Orientate gelatin blocks with a knife.
- Freeze blocks, and section at 10-20 µm using a microtome
- Transfer sections on to a drop of water on APS-coated slide.
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.