Carl fluorescence microscopy

Paraffin-embedded sections (4 μm) were performed, and followed by antigen retrieval. The sections were washed in phosphate-buffered saline and incubated with primary antibodies including anti-FUT4 (1:150) at 4 °C overnight, and followed by an incubation with the secondary antibodies including Alexa Fluor 594-conjugated Goat Anti-Rabbit IgG (1:300) (Proteintech, Wuhan, China) and FITC-LTL lectin (1:500) (Vector Laboratories, Burlingham, CA) at 37 °C for 1 h. The sections were then counterstained with 4, 6 diamidino-2 phenyl-indole (DAPI; Sigma-Aldrich, St. Louis, MO, USA) for nuclear staining. Images were taken by a Carl Zeiss fluorescence microscopy (Carl Zeiss, Hallbergmoos, Germany).
Cell immunofluorescence staining was conducted after fixing cells with 10% formaldehyde for 40 min, and permeabilized with 0.3% Triton X-100. 2% BSA was utilized to block the non-specific binding for 30 min at room temperature. Then cells were incubated with the primary antibodies and secondary antibodies as mentioned above.

Cells were transfected with miRNA mimic or miRNA inhibitor as described above and cultured in glass coverslips. Cells were then washed with PBS and subsequently fixed with cold 4% formaldehyde for 10 min at room temperature. Following fixation, cells were permeabilized with PBS containing 0.3% Triton X-100 and 2% BSA for 30 min at room temperature. Primary antibody Ki67 (Abcam, Cambridge, MA, USA) or ST8SIA4 was diluted 1 : 200 in 5% BSA in PBS and incubated on slides overnight at 4 °C. Slides were then washed three times with PBS and incubated 1 h with secondary antibody. After two washes with PBS, slides were incubated for 10 min with 4, 6-diamino-2-phenylindole (DAPI; Sigma-Aldrich, St. Louis, MO, USA) in PBS for nuclear staining. Images were taken in a Carl Zeiss fluorescence microscopy (Carl Zeiss, Hallbergmoos, Germany).