Anti c kit

Cross-sections of uterine horn samples were fixed in 4% PFA in 0.1 M PBS (pH 7.4) and cryoprotected in 18% sucrose. Immunostaining was carried out on consecutive 7 mm cryostat sections. To block endogenous peroxidase, the sections were treated with hydrogen peroxide in methanol and washed in 0.1 M PBS. The sections were blocked with 10% normal goat serum (Sigma, G9023) for 1 h at RT, incubated overnight at RT with a 1:100 dilution of anti-C-KIT (Dako, K1906, CA, USA), anti-OCT3/4 (Abcam, 19857), anti-NANOG (Abcam, 80892), or anti-SOX2 (Sigma, S9072) antibodies, washed in PBS, incubated for 1 h with a 1:25 000 dilution of biotinylated antirabbit (Vectastain ABC Kit; Vector Laboratories, PK 4001, Burlingame, CA, USA) antibodies, then washed, incubated for 45 min with the ABD reagent in PBS, and washed again. Proteins were visualized by incubating the sections in 0.3 mg/ml 3,30-diaminobenzidine tetrahydrochloride in 0.01% hydrogen peroxide in Tris-buffered saline (pH 7.2) for 2-3 min. Finally, the sections were dehydrated and coverslipped with the DPX mounting medium (Park Scientific Ltd, D-11601, Northampton, UK). To determine the specificity of the immunohistochemical staining, two controls were performed: first, the primary antibody was omitted during the