Vimentin antibody

The third generation of DPCs was plated on cover glass (WHB, China) in 6-well plates for culturing 4 days. Removed the basal medium and rinsed for three times using phosphate buffer solution (PBS). And then fixed with 4% paraformaldehyde (Solarbio, #P1110, China) for 30 min in room temperature and washed with PBS for three times, permeabilized with 0.5% Trixton X-100 for 15 min in room temperature and washed with PBS for three times, blocked with goat serum (BOSTER, #12C09A, China) for 30 min and incubated with α-SMA antibody (BOSTER, #BM0002, China) /or Vimentin antibody (BOSTER, #BM0135, China) /or Wnt10b antibody (orb97574, biorbyt, U.S.A.) overnight at 4°C in wet box [18–20 (link)]. For immunofluorescence staining, we used SABC-FITC SP kit (BOSTER, #SA1062, China). The DPCs were incubated with goat anti mouse IgG secondary antibody for 30 min at 37°C and then added SABC-FITC for incubating at 37°C in darkness for 30 min after washing with PBS. Counter-staining with DAPI and mounting with anti-fluorescence quenching agent (Beyotime, #P0128, China). The fluorescence signals were observed by using a fluorescence microscope (Nikon ECLIPSE 80i, Japan).

Total proteins from ZFPM2-AS1 shRNA lentivirus infected HCC cells were extracted using cell lysis buffer (BioFeng, Changsha, Hunan, China). After the protein concentrations were determined by BCA kits (TianLi Biotech, Ningbo, Zhejiang, China), 35 micrograms of proteins were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (8–12%), followed by transferring on to polyvinylidene difluoride membranes. Membranes were then blocked with 5% bovine serum albumin and subsequently immunoblotted at 4 °C for 10–12 h using primary antibodies: N-cadherin antibody (1:800; CST, Danvers, MA, USA); vimentin antibody (1:800; BOSTER, Wuhan, Hubei, China); GOLM1 antibody (1:1000; Abcam, Cambridge, UK); glyceraldehyde 3-phosphate dehydrogenase (abbreviated GAPDH) antibody (1:5000; PTG, Wuhan, Hubei, China). After incubating with secondary antibodies (2 h), the proteins were detected with ECL-Plus kits (Anhe Biotech, Qingdao, Jinan, China).