5 bromo 4 chloro 3 indolyl β d galactopyranoside

Biotinylated EGF for the EGFR was linked to biotinylated β-gal using fluorophore-conjugated SA. Ligand, linker, and reporter fragment were mixed in a molar ratio 1:1:3 at room temperature for 1 h. Excess d-biotin was added to block any remaining unbound SA sites. In the case of control assays, untargeted reporter complex was prepared with d-biotin, in place of the ligand, mixed with linker and reporter fragment in a molar ration 1:1:3. The ligand-complex was then diluted to 500 mL with cell feeding media (DMEM, 10% fetal bovine serum, 1% penicillin–streptomycin) and added directly to coverslips seeded with cells overexpressing human EGFR. Cells were incubated with EGF ligand-complex for 1 h at 37 °C. The cells were then fixed with 4% paraformaldehyde, rinsed with X-gal wash buffer, and stained overnight at 37 °C with 1 mg mL⁻¹ X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; Sigma; St Louis, MO, USA) in 5 mM K₃Fe(CN)₆, 5 mM K₄Fe(CN)₆, and 2 mM MgCl₂ in PBS. Cells were rinsed twice with PBS for 5 min. Images were captured by a Retiga EXi camera connected to a Leica DM4000 B upright microscope (Leica Microsystems; Wetzlar, Germany).

After fixation of mutant and control E13.5 embryos in 0.2% glutaraldehyde in PBS for 30 min at room temperature, embryos were washed three times in rinse solution (0.005% Nonidet P-40 and 0.01% sodium deoxycholate in PBS) and then stained in 5 mM potassium ferricyanide, 2 mM MgCl₂, 0.4%β-gal (5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside, B4252 Sigma Aldrich) in PBS for 2–3 h at room temperature followed by 2 times rinse in PBS and post-fixation in 3.7% formaldehyde. Frozen 20 µm sections were mounted on slides and fixed in 0.2% glutaraldehyde in PBS for 10 min on ice. Slides were incubated in detergent rinse solution for 10 min, then stained in β-gal staining solution at 37 °C for 2–3 h and post-fixed in 3.7% PFA for 2 h. Slides were counterstained with Nuclear Fast Red and Eosin solution.