Method
1.Dissect embryos in PBS
2.Immediately transfer the embryos to fixative at 4^oC, incubate in dark for 15 minutes
3.Rinse the embryos with rinse buffer at room temperature for three times, each 5 minutes
4.Incubate in dark with staining buffer at 37^oC overnight

Materials
10% NP40
1% Deoxycholate
0.5 M EGTA (make lM and dilute with 4 M NaOH till dissolves. Check pH = neutral - make up volume for 0.5 M)
0.5 M K₃Fe(CN)₆ = 1.65 g/10 ml (red)
0.5 M K₄Fe(CN)₆ = 2.10 g/10 ml (yellow)
40 mg/ml X-gal (in dimethylformamide)
1M MgCl₂

1.Fixative
4% Paraformaldehyde in PBS (dissolved at 65^oC)

2.Rinse buffer

Final concentration	Stock
5 mM EGTA	2.0 ml
0.1% Deoxycholate	2.0 ml
0.02% NP40	0.4 ml
2 mM MgCl2	0.4 ml
PBS	195.2 ml

3.Staining buffer

Final concentration	Stock
5 mM K3Fe(CN)6	0.4 ml
5 mM K4Fe(CN)6	0.4 ml
5 mM EGTA	0.4 ml
0.01% Deoxycholate	0.4 ml
0.02% NP4O	80 ul
2 mM MgCl2	80 ul
PBS	37.2 ml

Finally, add 1 ml of 40 mg/ml X-gal solution (in dimethyformamide) to the staining buffer slowly.

For RNA work, 5 ml of 4 M LiCl is added to 40 ml staining buffer, so that the LiCl concentration is ~0.5 M.