Akt c73h10

The sections were heated in a microwave oven in citrate buffer (pH =6.0) for 20 minutes at 95°C and cooled at room temperature. The sections were then washed with phosphate buffered saline (PBS) for three times (3 minutes/time). Endogenous peroxidase activity was blocked in 3% H₂O₂ for 20 minutes at room temperature. After washing with PBS for three times (3 minutes/time), sections were incubated with normal goat serum for 30 minutes at room temperature. The sections were then incubated overnight at 4°C with specificity primary antibody (anti-ERα [ab37438], Abcam, UK; anti-ERβ [ab3576], Abcam; PI3 kinase p110α [C73F8], Cell Signaling, USA; AKT [C73H10], Cell Signaling; E-cadherin [24E10], Cell Signaling; vimentin [D21H3], Cell Signaling) 37°C rewarming for 45 minutes. The sections were washed three times with PBS (3 minutes/time), then incubated with GTVision (Gene Technology Shanghai Co., Ltd., Shanghai, People’s Republic of China) for 30 minutes at 37°C. The sections were then washed three times with PBS and visualized with diaminnobenzidinetetra-hydrochloride (DAB kit; Shanghai Secco Chemical Technology Co., Ltd., People’s Republic of China). Finally, the sections were counterstained with hematoxylin and dehydrated.