CHAPTER 8 - HISTOLOGICAL METHODS
Agar Embedding for Cryostat Sectioning of Embryos or Larvae
(Source: R. BreMiller)
1. Fix animals as desired.
2. Wash in fix buffer, 3 times for 5 min each wash.
3. Melt an aliquot of agar-sucrose solution (1.5% agar, 5% sucrose) and cool to 4C.
4. Transfer an animal with a drop of fix buffer to a flexible plastic well. Remove excess fluid and add agar-sucrose to cover the animal. Use a fine wire or toothpick to move the animal around so that it is well coated and to orient it. Set aside until the agar has solidified (5 to 10 min).
5. Remove the block from the mold and trim with a razor blade on a hard surface according to the desired plane of sectioning. Leave at least 2-3 mm of agar around the tissue and 3-4 mm of agar below the tissue where it will be frozen to the cryostat chuck.
6. Place the trimmed agar block containing the animal into 30% sucrose and store at 4C until the block has sunk. For 1 to 2 day old fish this may be as short as a few hours.
7. When ready to section, make a slightly raised platform by freezing a layer of OCT compound (Tissue Tek, Miles) to the cryostat chuck. Add a drop of OCT to the platform, position the agar block and lower the whole unit slowly into a container of liquid nitrogen. Gradual even freezing is necessary to avoid cracking the block.
8. Equilibrate the block to the cryostat temperature before sectioning. Sections 10 to 40 µm thick can be cut with this procedure. Pick them up onto subbed slides and dry them on a 26C hot plate.
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