Goat anti lef1

Denatured protein extracted from skin tissue samples was loaded on a 10% SDS-PAGE gel, electrophoresed, and then transferred onto a PVDF membrane. PVDF membranes were blocked with 5% fat-free milk, and the membranes were then probed with rabbit anti-Wnt5a (1:1,000; Abcam, USA), goat anti-Frizzled (1:1,000, Santa Cruz, USA), rabbit anti-Dvl3 (1:1,000, Santa Cruz, USA), mouse anti-β-catenin (1:1,000; Santa Cruz, USA), goat anti-Lef1 (1:1,000, Santa Cruz, USA), and mouse anti-GAPDH antibody (1:500, Zhongshan Goldenbridge, China) at 4℃ overnight. Blots were then incubated with secondary antibody. Peroxidase activity on the membrane was visualized on X-ray film using the ECL western blotting detection system.

Immunohistochemical analysis was performed on paraffin-embedded sections from dorsal skin. Sections were incubated with primary antibodies: rabbit anti-Wnt5a (1:100, Santa Cruz, USA), mouse anti-β-catenin (1:100, Santa Cruz, USA), rabbit anti-Dsh (1:100; Santa Cruz, USA), goat anti-Ki67 (1:100, Abcam, UK), goat anti-lef1 (1:100, Santa Cruz, USA), mouse anti-AE15 (1:4, provided by TT Sun, New York University, USA). HRP-labeled secondary antibodies (Zhongshan Goldenbridge, China) were used. A DAB kit (Zhongshan Goldenbridge, China) was used to detect the expression of antigens according to the manufacturer's protocol.