- Origin of PGCs: Detection of LacZ
labelled primordial germ cells in grafted embryos.
- All staining is done at room temperature unless indicated.
- 1. Fix embryos for 5-10 minutes in 4% paraformaldehyde +
0.25% Gluteraldehyde.
- 2. Wash embryos 2 x 10 min with PBS.
- 3. Dehydrate and mount in polyester wax (mp 37 oC)
- 4. Section at 7um.
- 5. Dewax and hydrate slides through EtOH series.
- 6. Incubate in PBS for 10min.
- 7. Block non-specific binding for 20 min with 10% normal
Goat serum.
- 8. Incubate f
- or 60 min with rabbit IgG, anti-beta-galactosidase at 5ug/ml
(Cappel Cat. no 55976). Stock is 3mg/ml (this batch).
- 9. Wash 3 x 10 min with PBS + 0.2% BSA.
- 10. Incubate for 30 min with goat biotinylated anti-rabbit-IgG
(Vector Cat. No. BA1000), at 1:200 dilution.
- 11. Wash 3 x 10 min with PBS + 0.2% BSA.
- 12. Incubate slides for 60min with streptavidin-beta-gal
conjugate (Bohringer Mannheim Cat. No. 11112481), at 1:200 dilution.
- 13. Wash 2 x 10 min with PBS, then 1 x 10 min. with X-gal
washing buffer.
- 14. Incubate slides for 30-45 min at 37 oC with X-Gal staining
solution.
- 15. Wash for 5 min in ddH2O.
- 16. Mount in favourite substance.
- Dont be afraid to use another combination of 1o, 2o, or colour
development as your needs require.