Trilogy reagent

Tissues were fixed in formalin and embedded in paraffin, and 2-μm-thick consecutive sections were sliced and mounted on glass slides. The slides were first incubated at 65°C for 30 min and then subjected to deparaffinization in xylene followed by rehydration in a graded ethanol series. Then, the sections were boiled in Trilogy reagent (Cell Marque, Rocklin, CA, USA) for 10 minutes for antigen retrieval. After washing with 1 × PBS, the slides were immersed in 3% hydrogen peroxide for 10 min to suppress endogenous peroxidase activity. After three rinses with 1 × PBS, the sections were exposed to a mouse anti-vimentin antibody (Genetex, Irvine, CA, USA) for 1 hour at room temperature. After three rinses with 1 × PBS, the slides were incubated in a biotinylated secondary antibody (Dako, Glostrup, Denmark) for 25 min. The slides were then rinsed three times with 1 × PBS, followed by the addition of horseradish peroxidase (HRP)-conjugated streptavidin for 25 min at room temperature. Peroxidase activity was detected by incubating the slides in the chromogenic substrate 3, 3′-diaminobenzidine (DAB) (Dako) at room temperature. The slides were then counterstained with hematoxylin.

Tissues were fixed in formalin and embedded in paraffin, and 2-μm-thick consecutive sections were cut and mounted onto glass slides. The slides were first incubated at 65°C for 1 hour and then deparaffinized in xylene, rehydrated in graded ethanol solutions, and finally boiled in Trilogy reagent (Cell Marque, Rocklin, CA) for 10 minutes for antigen retrieval. After washing with 1× phosphate-buffered saline (PBS), the slides were immersed in 3% hydrogen peroxide for 10 minutes to suppress endogenous peroxidase activity. After three rinses with 1× PBS, the sections were subsequently incubated with antibodies against Hsp90, vimentin, N-cadherin and E-cadherin for 1 hour at room temperature. After three rinses with 1× PBS, the slides were incubated with a biotinylated secondary antibody (Dako, Glostrup, Denmark) for 25 minutes. Following three rinses with 1× PBS, horseradish-peroxidase conjugated streptavidin was added for 25 minutes at room temperature. Peroxidase activity was detected using 3,3-diaminobenzidine (DAB) (Dako) at room temperature. The slides were then counterstained with hematoxylin.

The tumors of the mice were fixed in formalin and embedded in paraffin. The 2-μm-thick consecutive sections were cut from the paraffin-embedded tissue blocks and floated onto glass slides. The slides were first incubated at 65 °C for 1 h and then deparaffinized in xylene, rehydrated in graded ethanol solutions, and boiled in Trilogy reagent (Cell Marque, Rocklin, CA, USA) for 10 min for antigen retrieval. After washing with 1 × PBS, the slides were immersed in 3% hydrogen peroxide for 10 min to suppress endogenous peroxidase activity. After triple-rinsing with 1 × PBS, the sections were exposed to the appropriate primary antibodies for 1 h at room temperature, after which they were rinsed three times with 1 × PBS and incubated with a biotinylated secondary antibody (Dako, Glostrup, Denmark) for 25 min. After three rinses with 1 × PBS, the slides were treated with horseradish peroxidase-conjugated streptavidin for 25 min. The peroxidase activity was developed with DAB (Dako), followed by counterstaining with hematoxylin.