Rabbit monoclonal anti hes1 antibody

Total protein was extracted from the human ESCC cells and mouse tissues with a standard method. Proteins were detected with a rabbit monoclonal anti-cleaved NOTCH1 antibody (1:500, Cat #: 4147, Cell Signaling Technology, Danvers, MA, USA), a rabbit monoclonal anti-PAX9 antibody (1:600, Cat #: 12847, Cell Signaling Technology), a rabbit monoclonal anti-HES1 antibody (1:4000, Cat #: 11988, Cell Signaling Technology), a rabbit monoclonal anti-RBPJ antibody (1:2000, Cat #: 5313, Cell Signaling Technology), a rabbit polyclonal anti-c-MYC antibody (1:1000, Cat #: 9402, Cell Signaling Technology), a mouse monoclonal anti-STAT3 antibody (1:1000, Cat #: 9139, Cell Signaling Technology), a rabbit polyclonal anti-p-STAT3 antibody (1:1000, Cat #: 9131, Cell Signaling Technology), a rabbit polyclonal anti-SOX2 antibody (1:2000, Cat #: ab97959, Abcam, Cambridge, MA, USA), a rabbit polyclonal anti-ETV4 antibody (1:1000, Cat #: ab135590, Abcam) and a mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:40000, Cat #: ab8245, Abcam).

For IHC staining, deparaffinized sections were pretreated to retrieve antigens with a Tris-based Antigen Unmasking Solution (Vector Laboratories, Burlingame, CA, USA), before blocking with 10% normal serum and then applying either a rabbit monoclonal anti-NOTCH1 antibody (1:200, Cat #: D1E11, Cell Signaling Technology), a rabbit polyclonal anti-NOTCH2 antibody (1:100, Cat #: ab52302, Abcam), a rabbit polyclonal anti-activated NOTCH1 antibody (1:100, Cat #: ab8925, Abcam), a rabbit monoclonal anti-PAX9 antibody (1:50, Cell Signaling Technology) or a rabbit monoclonal anti-HES1 antibody (1:50, Cell Signaling Technology) at 4°C for overnight. Tissue sections were then washed in PBS and incubated with biotinylated secondary antibodies for 30 minutes at room temperature. Detection of the antibody complex was done using the streptavidin-peroxidase reaction kit with DAB as a chromogen (ABC kit, Vector Laboratories).
NICD1 IHC staining intensity in histologically normal esophageal squamous epithelium was measured with ImageJ. The region of the esophageal epithelium was marked for analysis of the mean density of brown staining. The staining intensity was calculated by subtracting the mean intensity of the background.