The tumor tissues were selected, dehydrated using gradient alcohol,and soaked in 3% H2O2 for 10 min. After a wash under distilled water and antigen repair, the tissues were boiled in the 0.01 M citric acid buffer solution (pH = 6.0) at 95°C for 15–20 min. After natural cooling for 20 min to room temperature, the tissues were washed by PBS three times, each time for 5 min. Then 100 μl 5% BSA blocking liquid was added and incubated at 37°C for 30 min. Then the diluted
primary rabbit monoclonal antibodies, including E‐cadherin (ab40772, 1:500; Abcam Inc.), vimentin (ab92547, 1:200; Abcam Inc.), N‐cadherin (ab18203, 1 ug/ml; Abcam Inc.), snail (1:1000, ab180714; Abcam), and twist (1:1000, ab49254; Abcam) were added for overnight incubation at 4°C. After washing with PBS three times, each time for 3 min, the biotin‐labeled secondary goat antirabbit antibody (HY90046, 1:100; Shanghai Hengyuan Biological Technology Co. Ltd., Shanghai, China) was added for incubation at 37°C for 30 min.
