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 ||L gel albumin with 75 ||L glutaraldehyde, and quickly pour over the embryo.

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

6. Sections are cut at 50 |im 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 |im 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.

Was this article helpful?

0 0

Post a comment