Mouse anti vim

Immunoblot analysis was performed as previously described [23 (link)], using rabbit anti-TFF3 antibody [14 (link)]. Mouse anti-β-ACTIN, rabbit anti-p-c-SRC, mouse anti-c-SRC, and mouse anti-γ-CTNNG antibody was obtained from Santa Cruz Biotechnology, Santa Cruz, CA, USA. Mouse anti-CDH1, mouse anti-CDH2, rabbit anti-OCLN, mouse anti-VIM, mouse anti- ITGA6, rabbit anti-pSTAT3, and mouse anti-STAT3 antibody was obtained from Abcam, Cambridge, MA, USA. Cell extracts were resolved by SDS-PAGE and immunoblotted, with the respective antibodies, as previously described [23 (link)]. β-ACTIN was used as input control for cell lysate. The sizes of detected protein bands in kDa are shown on the left side.
Confocal laser scanning microscopy was performed as previously described [22 (link)]. Nuclei were visualised with VECTASHIELD mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA). Images were captured using a confocal microscope (Eclipse C1 Plus Confocal; Nikon Instrument Inc., Melville, NY, USA). Primary antibody, Mouse anti-CDH1, and mouse anti-VIM was obtained from Abcam. Secondary antibody, Alexa Fluor 488 rabbit anti-mouse immunoglobulin (Ig)G and Alexa Fluor 568 rabbit anti-mouse IgG was obtained from Invitrogen, Singapore. Rhodamine-conjugated phalloidin (Sigma-Aldrich, St Louis, MO, USA) was used to visualize f-actin filaments.

Mouse liver tissues were formalin fixed, embedded in paraffin and sectioned at 4 μm. Slides were routinely processed for immunohistochemically stained using the following antibodies: rabbit anti-αSMA (1:200, Abcam, MA, USA), rabbit anti- FLNA (1:1000, Abcam, MA, USA), mouse anti-VIM (1:500, Abcam, MA, USA). Visualization was perfomed using the Histostain-Plus Kit (ZSGB-BIO). For immunofluorescence studies, the isolated HSCs were cultured in DMEM/F12 supplemented with 10% fetal bovine serum for 1 month and kept stable proliferation rate and growth characteristics. These primary activated HSCs grown on cover slips were fixed and stained using the above antibodies. 20x pictures for each slip was analyzed using Image J software. The detail of mice liver fibrosis model and isolation of mice HSCs are provided in the supporting information.

Tumor cells on coverslips were fixed with 4% paraformaldehyde for 15min. Then, in PBS containing 10% bovine serum albumin, the sections were blocked at room temperature for 2 h. After blocking, samples were incubated with primary antibodies specific for mouse anti-E-cad (Abcam, Cambridge, UK), mouse anti-Vim (Abcam, Cambridge, UK) overnight at 4℃. Fluorescent secondary antibody was carried out for 1 h at room temperature. Cell nuclei were counterstained with DAPI (Sigma-Aldrich, MO, USA). Images were acquired on a Zeiss LSM510 confocal microscope (Oberkochen, Germany).