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David M. Ornitz, M.D., Ph.D.

 

LacZ-cell Staining

Materials:

1) 0.5% glutaraldehyde in PBS
2) 20mM K4Fe(CN)6o3H2O 0.44g into 50 ml PBS
3) 20mM K3Fe(CN)6 0.33g into 50 ml PBS
4) X-gal 2% in DMF
5) PBS containing 1mM MgCl2
6) Staining solution

Ingredients: Per 1 ml:
20 mM K4Fe(CN)6o3H2O/PBS 250 l
20 mM K3Fe(CN)6/PBS 250 l
1 M MgCl2 1 l
2% X-gal 50 l
PBS to 1 ml

Procedures:

1) Wash the cells twice with 2 ml PBS (I'm assuming you're working with a 35 mm dish, otherwise, adjust your volume accordingly).
2) Fix the cells for 10 minutes at room temperature in 2ml of 0.5% glutaraldehyde diluted in PBS.
3) Wash the cells 3 times with 2 ml PBS containing 1 mM MgCl2.
4) Cover the cells with staining solution and incubate at 37C until the desired staining is achieved.
5) Wash cells with PBS.


β-galactosidase Embryo Staining Protocol

1. Prepare fix as follows: weigh out 0.5 g of solid paraformaldehyde into a 50 ml conical tube. To this tube add:

5ml 0.5M Pipes buffer (pH 6.9)
50l 1M MgC12
125l 0.2 M EGTA(pH 8.0)

Heat in microwave for 5 second intervals until PFA dissolves completely. Be very careful not to overheat the tube (IT WILL EXPLODE) and always release pressure by unscrewing the tube in between heatings.

2. Embryos are first dissected away from the placental material and the amnion is removed. When dissecting many embryos at once, the initial embryos can be stored in 1XPBS while other embryos are dissected. Prior to fixation, the embryos are very delicate so exercise care in handling. A cut off P1000 pipet tip works well for moving embryos around.

3. Once all the embryos are dissected, wash them 1 or 2 times in 1XPBS. Then fix the embryos by placing them in the PFA solution for 15 minutes. Although fixation tends to harden the embryos, one should still exercise care when manipulating the embryos.

4. After fixing the embryos, wash them 3 times @ 5 minutes per wash in 1XPBS/1 mM MgCl2.

5. Prepare staining solution as follows, mix:

250l 20 mM K3Fe(CN)6/PBS
250l 20mM K4FE(CN)6o3H20/PBS
1l 1M MgCl2
1l 10% sodium desoxycholate
2.2l 9% NP40
50l 2% Xgal

and bring up to a final volume of 1.05 mls with 1XPBS. One can also add detergents to the staining solution if desired, add This may help to increase the permeability of the embryo during staining.

6. Place embryos in the staining solution and incubate at 30-37C overnight.

7. After desired degree of staining is achieved, wash embryos in 1XPBS. Embryos may then be post-fixed or stored directly in 1XPBS. 10XPBS (pH 7.2) 1L_ NaC1 80.06g 1.37M KC1 2.01 g 27 mM Na2HPO4 11.35g 80 mM KH2PO4 3.53 g 26 mM 20 mM K4 Fe(CN)6o3H20 Mix 0.44221 g solid 5 ml 10XPBS Bring volume up to 50 ml with water. 20 mM K3 Fe(CN)6 Mix 0.32926 gsolid 5 ml 10XPBS Bring volume up to 50 ml with water X-gal is made 2mg/ml or 2% in DMF.