Bsa 5

CRC tissues were fixed in 4% formaldehyde solution for 2 h at 25°C and then sectioned into 5-µM-thick frozen sections. The sections were washed in cold PBS 3 times and subsequently blocked with 2% bovine serum albumin V at 25°C (BSA-V; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) for 1 h. Samples were incubated with primary antibodies against E-cadherin (1:20; sc-8426; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) diluted with 1% BSA-V overnight at 4°C. Following 3 washes with PBS, the sections were incubated with tetramethylrhodamine conjugated goat anti-rabbit secondary antibody (1:1,000; sc-362281; Santa Cruz Biotechnology, Inc.) diluted with 1% BSA-V in the dark for 1 h at 25°C and washed in PBS again for 3 times. DAPI diluted with PBS was used to stain the nuclei at 25°C. Images at a magnification of ×40 were captured using an inverted fluorescence microscope (Nikon Corp., Tokyo, Japan).