Notch3

Apical papillae were obtained as mentioned above and processed for cryosectioning; 8 μm-thick sections were fixed with cold acetone at −20 °C for 15 min, washed in PBS, treated with 1.5% hydrogen peroxide for 30 min and blocked with 2.5% normal horse serum (Vectastain Elite ABC kit; Vector laboratories) for 1 h. Tissue sections were then incubated with mouse monoclonal anti-NOTCH3 antibodies (dilution 1:100, Abcam, USA) for 1 h at room temperature, followed by washing and incubation with biotinylated anti-mouse immunoglobulin G (secondary antibody) for another 1 h. After washing, avidin-peroxidase complex was added and incubated for 30 min, followed by washing and the addition of peroxidase substrate solution for 5 min. Sections were counterstained with hematoxylin solution (Sigma, USA). Negative control slides were prepared in parallel without adding the primary antibody.
For immunofluorescence staining, frozen sections of AP were fixed with cold acetone at −20 °C for 15 min, washed and blocked for 1 h. Sections were then incubated with primary antibodies: NOTCH3 (dilution 1:100, Abcam, USA) and CD146 (dilution 1:50, Invitrogen, USA) for 1 h at room temperature, followed by washing and incubation with the appropriate fluorophore-conjugated secondary antibodies for another 1 h.