1)  On Frozen sections

a. X-Gal Staining of Embryos frozen sections or Tibia

Day 16.5/17.5 embryos were frozen on dry ice in tissue freezing medium (Leica) and cut into 15 µm sections. Tibiae were cut longitudinally. After fixation in 4% PFA for 5 min, specimen were incubated 24 hr in PBS containing 5 mM K₃[Fe(CN)₆], 5 mM K₄[Fe(CN)₆], 2 mM MgCl₂, and 1mg/ml X-Gal at 37°C.

2)  On Whole mount

 a. Whole mount X-gal(LacZ), PECAM-1, and Histological Staining

Embryos and yolk sacs were removed between E7.5 and E10.0, fixed in cold 4% paraformaldehyde/PBS for 10 min, rinsed twice with PBS, and stained for 1 hr to overnight at 37°C in X-Gal buffer (1.3 mg/ml potassium ferrocyanide, 1 mg/ml potassium ferricyanide, 0.2% Triton X-100, 1 mM MgCl₂, and 1 mg/ml X-Gal in PBS [pH 7.2]). LacZ-stained embryos were post-fixed and photographed or sectioned on a cryostat after embedding in 15% sucrose and 7.5% gelatin in PBS. Procedures for whole-mount or section staining with anti-PECAM-1 antibody (clone MEC 13.3, Pharmingen) were done essentially as described (Fong et al., 1995 ; Ma et al., 1998 ). HRP-conjugated secondary antibodies (Jackson) were used for all PECAM-1 stainings except for Figure 4, where alkaline phosphatase was the enzyme of choice. LacZ-stained yolk sacs were sectioned in gelatin and then subjected to hematoxylin counterstaining by standard procedures.

Cell, Vol. 93, 741–753, May, 1998

b. Nuk- gal Staining

For whole-mount staining, embryos were collected in 0.1 M PBS (pH 7.3), incubated at room temperature for 30 min in fresh lacZ fix buffer (0.2% gluteraldehyde, 5 mM EGTA, 2 mM MgCl₂ in PBS), rinsed several times in wash buffer (2 mM MgCl₂, 0.02% NP-40 in PBS), and incubated at 37°C overnight in lacZ staining buffer (wash buffer containing 1 mg/ml X-gal, 2.12 mg/ml potassium ferrocyanide, and 1.64 mg/ml potassium ferricyanide). Embryos were then rinsed in wash buffer, postfixed in formalin, dehydrated in an ethanol series, and cleared in benzyl alcohol:benzyl benzoate (1:2) immediately prior to observation and photography. For histochemical analysis, embryos were dehydrated in ethanol, embedded in paraffin, sectioned at 6 µm, and counterstained with nuclear-fast red.

c. LacZ Staining

E11.5 embryos were dissected, fixed for 30 min at room temperature in 4% PFA in PBS, washed twice with PBS, and incubated in Xgal staining solution. An analogous staining procedure was employed to determine the colonic expression pattern in adult mice, except that the staining was performed at room temperature and 20 mM Tris (pH 7.3) was added as described (Hogan et al., 1994 ).