Peroxidase blocking reagent

Organotypic raft sections, obtained as above, were deparaffinized in xylene and re‐hydrated through graded ethanols to PBS, pH 7.4. Antigen retrieval was achieved by heating sections in target retrieval solution high pH (Dako, Carpinteria, CA) for 15 min at 97°C and endogenous peroxidase activities were blocked by peroxidase blocking reagent (Dako) for 10 min at 25°C. Sections were then washed with PBS and probed with rabbit polyclonal anti‐K1 (1:500 in PBS, AF 87, Covance, Princeton, NJ) for 1 h in a humidified chamber. Slides were washed extensively in PBS and detection was performed using an HRP‐conjugated secondary antibody (Dako) followed by colorimetric detection using DAB substrate chromogen (Dako) for 5 min. Sections were counterstained with hematoxylin, dehydrated with ethanol and xylene and permanently mounted under a coverslip.

Sectioned tumour slides were deparaffinized and rehydrated. The sections were
incubated in antigen retrieval (pH 9.0), washed with TBST washing buffer and
incubated with Dako^® peroxidase blocking reagent for 1 h. The
sections were then washed with TBST (three times, 5 min per wash). The slides
were blocked using blocking solution (10% goat serum and 5% BSA in TBST) for
1 h. Slides were then washed again and incubated with primary antibodies against
Ki67 (Dako, Clone MIB-1, Cat#M7240, mouse monoclonal) overnight at 4°C.
Subsequently, the slides were washed and incubated with biotinylated secondary
antibody for 1 h, followed by washing with TBST (three times, 5 min each) to
remove unbounded antibodies. The sections were incubated with ABC solution for
1 h, and remaining solutions were removed through washing with TBST as
previously described. Finally, 200 µl of Dako^® DAB solution was
applied to each section, and samples were monitored closely to examine the
development of stains. The slides were counterstained with haematoxylin, washed,
left to dry overnight and mounted. Slides were imaged using a light microscope
(CX41, Olympus) at 100× magnification. The ratio of stained cells
versus total cells were scored using Image J software.

The following antibodies were used in this study: anti-ZEB1 (E-20), anti-vmentin (V9), anti-E-cadherin (H-108), anti-PDGFRα (C-20), anti-p-PDGFRα (Y754) and anti-β-actin (I-19) antibodies (Santa Cruz Biotechnology, Dallas, TX, USA); an anti-SHP-2 antibody (no. 610621; BD Biosciences, San Jose, CA, USA); anti-ZEB1 (D80D3), anti-p-p44/42 MAP kinase (Thr202/Tyr204, no. 9101), rabbit anti-p44/42 MAP kinase (no. 9102), anti-p-Akt (S473, no. 4060) and anti-Akt (no. 9272) antibodies (Cell Signaling Technology, Danvers, MA, USA). The secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Peroxidase blocking reagent was from DAKO (Carpinteria, CA, USA); AquaBlock was from East Coast Biologics Inc. (North Berwick, ME, USA). Inhibitors, PD98059, PHPS-1 and LY294002 were from Sigma (St. Louis, MO, USA). Cell culture media and other reagents were from Invitrogen (Carlsbad, CA, USA), Sigma-Aldrich or Peprotech (Rocky Hill, NJ, USA).