Pk90017s

For immunostaining, frozen sections of skin tissues were fixed with acetone for 30 min and permeabilized with 0.1% Triton-X100 for 5 min. Frozen sections were blocked with 5% BSA for 60 min and incubated with anti-SOSTDC1 (PK90017S, Abmart, Shanghai, China) antibody (1:150) at 4 °C. On the second day, frozen sections were incubated with Cy3-labeled goat anti-rabbit IgG (A0516, Beyotime, Shanghai, China) (1:300) for 60 min in the dark. Nuclei were stained with DAPI (C1005, Beyotime, Shanghai, China). The photographs were taken using a fluorescence microscope (RVL-100-G, ECHO, San Diego, CA, USA).

One-month-old ALC, Tan, Ujimqin, MF and Aohan fine wool lambs were used for total protein extraction and subsequent Western-blot. In these five lamb breeds, 3 of each were selected for mixing pools, where Merino-1 and Merino-2 were derived from mixing pools with 3 Aohan fine wool lambs skin tissue, respectively. The skin tissues of these lambs were well ground in liquid nitrogen and the total proteins were extracted by IP lysate (P0013, Beyotime, Shanghai, China). The total proteins and the pre-stained protein molecular weight marker (MF291-01, Mei5bio, Beijing, China) were separated by 10% SDS-PAGE and transferred onto nitrocellulose membrane, then blocked one hour with blocking solution (P0252, Beyotime, Shanghai, China). The membrane was incubated overnight with mixed antibodies, that is SOSTDC1 (PK90017S, Abmart, Shanghai, China) antibody with 1:1,000 dilution and β-actin antibody with 1:2,000 dilution (AF5003, Beyotime, Shanghai, China). The overnight membrane was washed three times with washing solution (P0023C, Beyotime, Shanghai, China), then the membrane was incubated with a 1:2,000 dilution of HRP conjugated anti-rabbit secondary antibody (A0208, Beyotime, Shanghai, China) for 1 h at room temperature. Target protein were detected by Gel Documentation System (G:box, SynGene, Cambridge, UK). Band density was analyzed using image J software.