Vimentin xp rabbit mab

Cells were fixed in 2% PFA, permeabilized with 0.5% TritonX-100 at 4° for 10 min, and blocked with PBS containing 5.0% (v/v) normal goat serum and 0.3% (v/v) Triton X-100. Immunofluorescence staining was performed with mouse mAb Ki-67 (Dako, Hamburg, Germany), Vimentin XP Rabbit mAb, E-cadherin rabbit mAb and Zona Occludens mAb (Cell Signaling Technologies, MA, USA), and appropriate secondary antibodies (goat-anti-mouse IgG-Alexa488 and goat-anti-rabbit IgG-Alexa467, Invitrogen, Karlsruhe, Germany). The slides were then mounted with Prolong Gold Antifade Reagent with DAPI (Invitrogen Life Technologies, Carlsbad, CA, USA).

The total protein was extracted using RIPA buffer (Beyotime Institute of Biotechnology, China) mixed with a phosphatase inhibitor and protease inhibitor (CWBIO, China). Equal amounts of protein (20–40 µg) were separated via SDS‒PAGE and transferred onto PVDF membranes (Millipore, MA). After using 5% milk blocking for 1 h, the membranes were placed with antibodies targeting GAPDH monoclonal antibody 1:60000 (#60004-1-lg, Proteintech, China), GLUT3 polyclonal antibody 1:5000 (#20403-1-AP, Proteintech, China), E-Cadherin Mouse mAb 1:1000 (#14,472, Cell Signaling Technology, USA), ZEB1 polyclonal antibody 1:1000 (#21544-1-AP, Proteintech, China), N-cadherin XP Rabbit mAb 1:1000 (#13,116,Cell Signaling Technology, USA), Vimentin XP Rabbit mAb 1:1000 (#5741, Cell Signaling Technology, USA), and NF-κB XP Rabbit mAb 1:1000 (#8242, Cell Signaling Technology, USA) at 4 °C overnight. The membrane was then incubated with the secondary antibodies 1:3000 (Beyotime). The visualisation of antibodies was completed through the ECL system.