2.1.Immunohistochemistry:
All tumor specimens were fixed in 10% buffered formalin and embedded in
parafin according to standard procedures. Serial 5µm- thick sections
were placed on positively charged slides. Immunohistochemical (IHC)
detection of E- cadherin (Mouse mAb clone NCH-38, IR05961-2, DAKO), β-
catenin (Mouse mAb clone beta-cat-1, IR70261-2, DAKO), N- cadherin
(Mouse mAB clone D-4, sc-8424, Santa Cruz, CA, USA), smooth muscle actin
(Mouse mAb, Clone 1A4, DAKO), and Zeb1 (Rabbit pAb, ab87280, ing ABCAM)
were performed. All IHC stainings were performed by an autostainer Link
AS48 (DAKO, Denmark) which uses the envision flex system. The sections
were observed and photographed using an Nicon Eclipse Ni microscope
equipped with a Nicon digital sight DS-U3 camera.