Anti e cadherin

Immunohistochemical staining was performed using Biotin-Streptavidin horseradish peroxidase (HRP) Detection Systems (SP-9000, ZSGB-BIO, Beijing, China). The tissue sections were incubated with 3% H₂O₂ deionized water for 10 min and blocked with goat serum (SP-9000, Reagent A) at room temperature. Then, the tissue sections were incubated with rabbit polyclonal anti-KLF4 (1:2,000 dilution, Cat. No. ab215036; San Francisco, Abcam), rabbit polyclonal anti-E-cadherin (1:400 dilution, Cat. No. 3195T; Danvers, CST), rabbit polyclonal anti-vimentin (1:3,000 dilution, Cat. No. 10366-1-AP; Rosemont, Proteintech) primary antibodies overnight at 4°C.
Immunofluorescence staining was performed to detect KLF4, E-cadherin, and vimentin expression. The tissue sections were blocked with 5% bovine serum albumin (BSA) and incubated with rabbit monoclonal anti-KLF4 (1:500 dilution, Cat. No. ab214666; San Francisco, Abcam), rabbit polyclonal anti-E-cadherin (1:200 dilution, Cat. No. 3195T; Danvers, CST), and rabbit monoclonal anti-vimentin (1:400 dilution, Cat. No. ab92547; San Francisco, Abcam) primary antibody overnight at 4°C separately. The tissue sections were incubated with GoatAnti-Rabbit IgG H&L(HRP) (1:300 dilution, Cat. No. ab6721; San Francisco, Abcam) second antibody at room temperature for 1 h.

Western blot analysis was conducted as previously described [24 (link)]. The following primary immunoblotting antibodies were used: anti-GAPDH, anti-E-cadherin, anti-Fibronectin, anti-Vimentin, anti-Snail, and anti-c-Met (Epitomics, Burlingame, CA, USA), anti-β-catenin (Proteintech, Chicago, USA), anti-N-cadherin, anti-c-myc, anti-EGFR, anti-p-EGFR (Tyr1068), anti-GSK3β, anti-p-GSK3β (Ser9), anti-AKT (pan), anti-p-AKT (Ser473), anti-stat3, anti-p-stat3 (Tyr705) and anti-CREB1 (Cell Signaling Technology, Beverly, MA).

SW480 and HCT116 cells were seeded on 12 mm round coverslides. After reaching appropriate densities, for EMT either as confluent or sparsely growing [19] (link), and as confluent growing cultures for CoCl₂ treatment (3 h, 100 µM CoCl₂, serum free) [21] (link), cells were fixed in the presence of 2% (v/v) formaldehyde. The following antibodies were employed: anti-β-catenin (1∶150; clone 14/Beta-Catenin, BD Biosciences, NJ, USA) and anti-E-cadherin (1∶100; clone EP6; Epitomics Burlingame, UK). Antibodies were diluted in 0.1% (w/v) saponin, counterstained with anti-Alexa-Fluor®488 (Invitrogen, Karlsruhe, Germany) and finally mounted in mounting medium containing DAPI (Vector Vectrashield Laboratories, Dossenheim, Germany). Immunofluorescence staining was detected by immunofluorescence-microscopy (Zeiss Axioscope 50, Zeiss Axiocam MRc).