PTEN was scored semiquantitatively using the immunoreactive score (IRS).
Immunoreactive signals were visualized using the DAB substrate, which stains the target protein a brown color.
The immunoreactive signals were detected using the Western Chemiluminescent HRP substrate kit (Millipore, Billerica, USA).
The immunoreactive proteins were visualized using the Metal Enchanced DAB Substrate Kit (Thermo Scientific) according to the manufacturer's instructions.
Peroxydase-conjugated secondary antibodies were used and immunoreactive bands were visualised using the West Pico chemoluminescent substrate (Perbio, Brebières, France).
Immunoreactive bands were visualized using the SuperSignal Femto Chemiluminescent Substrate (Pierce) in the ImageQuant RT ECL imaging system (GE Healthcare).
After incubation with the appropriate HRP (horseradish peroxidase -conjugated secondary antibodies (Amersham Biosciences), immunoreactive peroxidase -conjugateded by chemiluminesecondarying the SuperSignantibodieso chemiluminescent substrAmershamrce).
Immunoreactive proteins were detected by enhanced chemiluminescence using the SuperSignal West Pico substrate (Pierce Biotechnology, Rockford, IL) and HyBlot CL autoradiography film (Denville Scientific, Metuchen, NJ).
Next, the horseradish peroxidase (HRP -conjugated goat anti-rabbit IgG secondary antibody (12-348; Millipore) was applied and immunoreactive bands were visualized by enHRP -conjugatedinescence usingoate Luminata Forte HRP Substrate (Millipore) and detected wIgG the G:Box secondaryyngene).
CK7 and CK20 expression was established using the percentage of immunoreactive cells calculated by the number of immunoreactive cells over the total number of tumor cells.
The immunoreactive bands were detected using the SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Rockford, IL) according to manufacturer's directions.
