Preparation of Material for Sectioning

1. Dissect embryos in IX PBS (mice) or Howard's Ringer (chicks). Fix in a large volume (at least five times the volume of embryonic tissue) of 4% (w/v) parafomaldehyde in PBS at 4°C overnight in a vessel that adequately contains the fumes (see Subheading 2.3.). Smaller embryos can be fixed for shorter periods of time (4 h minimum), and it is convenient to dechorionate zebrafish after fixation. Embryos can be fixed pinned out in Petri dishes, if required, in the designated refrigerator. Fixation times will vary with the size of the embryo from a few hours (mice up to embryonic [E] 9.5, chicks to stage 18) to 12 h (E18 mice) or even longer (newborn mice or chicks from stage 38; see Note 1). In our experience fixation for 2-3 d does not adversely affect results.

2. Incubate in 1X PBS on ice for 30 min (small embryos, i.e., chicks <E6.5, mice <E13.5) or 1 h (larger material). Use glass containers, since later treatments involve high temperatures and organic solvents.

3. Further dissect embryos if required, e.g., cut embryos away from surrounding membranes. Stage according to Theiler (6) or Hamburger and Hamilton (7) if required.

4. Remove PBS and replace with 1X saline. Incubate for 30 min (small embryos) or 1 h (larger material) on ice.

5. Replace saline with saline:ethanol (1:1), and incubate for 30 min (small embryos) or 1 h (larger material) on ice.

6. Replace saline:ethanol with 70% (v/v) ethanol (prepared with DEPC-treated water), and incubate for 30 min (small embryos) or 1 h (larger material) on ice.

7. Repeat step 6.

8. Replace 70% (v/v) ethanol with 80% (v/v) ethanol and incubate on ice 30 min.

9. Replace with 95% (v/v) ethanol, and incubate on ice for 30 min (small embryos) or 1 h (larger material) on ice.

10. Replace with 100% ethanol, and incubate on ice for 30 min (small embryos) or 1 h (larger material) on ice (see Note 2).

11. Place embryos in 100% ethanol for 30 min (small embryos) or 1 h (older embryos).

12. Replace with 100% ethanol for 30 min (small embryos) or 2 h (older embryos).

13. Replace 100% ethanol with xylene or (preferably) histoclear in glass bottles, and incubate on ice 30 min (small embryos) or 1 h (older embryos).

14. Repeat step 13 with fresh xylene or histoclear.

15. Pour off and half-fill bottles with fresh molten wax (sections cut from blocks prepared with freshly melted wax have better elasticity than from wax that has remained molten for days), and place at 52°C for 45 min (small embryos) or 90 min (large embryos).

16. Change wax twice, and incubate each time for 60 min (small embryos) or 90 min (large embryos).

17. Embed tissue: fill molds with wax first (if possible, do this on a warmed plate to keep the wax molten), carefully transfer the embryo to the mold, orient with a hot mounted needle or warm forceps, and move to a cooled plate or onto the bench. Label one end of the block for reference after the wax is set and the embryo's orienation is no longer visible (e.g., by inserting a piece of card on which details of the specimen and its orientation are written) and allow the wax to set completely (1-2 h).

18. Blocks may be stored at 4°C for several years. Any degradation of RNA in such material is most likely owing to incomplete penetration of reagents during the above processing steps.

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