Sectioning After In Situ Hybridization

Two procedures are described: the first is a rapid method for producing thick sections, and the second is a method for producing thin sections as permanent mounts.

3.7.1. Thick Sections

1. Equilibrate embryos PBS; use several changes of PBS over 3-4 h.

2. Embryos are embedded in gel albumin. Thaw an aliquot of gel albumin, put 750 |L in a suitable mould, and mix with 75 |L glutaraldehyde (adjust volumes if required for larger embryos), and wait until a skin forms on top (a few minutes).

3. Put embryo (in a small volume of PBS) on top of setting gel albumin, suck off excess PBS, and orient the embryo as desired.

4. Mix separately 750 pL gel albumin with 75 pL glutaraldehyde, and quickly pour over the embryo.

5. Leave overnight at 4°C to set completely.

6. Sections are cut at 50 pm on a vibrotome, are mounted on slides in glycerol, coverslips are sealed with nail varnish and the preparations are stored at 4°C.

3.7.2. Thin Sections

The times indicated are for smaller embryos (e.g., stage 20 chicks or E10.5 mice) and should be increased for larger material.

1. Embryos are postfixed in 4% (w/v) paraformaldehyde in PBS for 4 h or overnight.

2. Place in glass vials, and dehydrate in 100% methanol for 5 min.

3. Remove the methanol, and equilibrate in propan-2-ol for 10 min.

4. Remove the alcohol, and clear in tetrahydronapthalene in a fume cupboard for 30 min.

5. Equilibrate in four changes of wax each for 1 h at 58°C.

6. Embryos are then embedded in wax in suitable moulds.

7. Sections are cut at a thickness of 10-20 pm and floated in a water bath.

8. Strips of 5-15 sections are collected onto gelatin- or polylysine-coated slides and allowed to dry. Then they are dewaxed in xylene (two changes each for 15 min) and mounted in DPX.

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