Anti vimentin rabbit polyclonal antibody

STEC grown on coverslips were washed three times with PBS after different treatments. The cells were fixed in cold methanol for 20 min and blocked with 3% BSA for 2 h. Cells were incubated with anti-vimentin rabbit polyclonal antibody (1:400, Proteintech, Wuhan, China) at 4 °C overnight and then with DyLight 488-conjugated goat anti-rabbit IgG antibody (1:400, Abbkine, Wuhan, China) at RT for 1 h. The nuclei were stained with 4',6-Diamidino-2-phenylindole (DAPI, Beyotime, Nanjing, China). The coverslips were mounted using ProLong Gold antifade reagent (Invitrogen, Carlsbad, CA, USA) and imaged with a fluorescence microscope (Axio Observer 7; LD Plan-NEOFLUAR 40 × /0.6; ZEISS, Germany).

The protein expression levels were assessed using western blot analysis. In brief, total cell lysates from different experiments were obtained by lysing the cells in RIPA buffer. The total cell lysates were separated on SDS-PAGE gels, transferred to PVDF membranes (Millipore), immunoblotted with antibodies [anti-snail rabbit polyclonal antibody, anti-ZO-1 rabbit polyclonal antibody, anti-β catenin rabbit polyclonal antibody, anti-vimentin rabbit polyclonal antibody, anti-fibronectin rabbit polyclonal antibody, anti-caveolin-1 rabbit polyclonal antibody, (all antibodies supplied by Proteintech, Wuhan, China), and anti-GAPDH monoclonal antibody (Bioworld, Nanjing, China)] and visualized using an enhanced chemiluminescence detection system (Amersham Biosciences, Chalfont St Giles, UK). The protein bands were quantitated by densitometry using gel analysis software ImageJ. The values were normalized to GAPDH expression.