Anti phospho p42 44 mapk thr202 tyr204

Tumor tissues were fixed in formalin, embedded in paraffin, sectioned to 5 μm, and mounted on slides. The sections were deparaffinized in xylene, rehydrated in graded alcohols, and washed in distilled water. Antigens on sections were retrieved by boiling in 10 mM citric acid (pH 6.0) for 40 minutes. Endogenous peroxidases were quenched by incubation in 3% H₂O₂ for 10 minutes at room temperature. The slides were washed 3 times with PBS and blocked for 30 minutes with 10% normal goat serum in 1% bovine serum albumin/PBS. The slides were then incubated with the following antibodies: anti-Ki-67 (Lab Vision), anti-phospho-p42/44 MAPK (Thr202/Tyr204) (Cell Signaling), anti-fibronectin (BD Transduction), anti-vimentin (Cell Signaling), anti-E-cadherin (BD Transduction), and anti-β-catenin (Cell Signaling). Stained slides were visualized with an Eclipse 80i microscope (Nikon), and the images were captured at a 200× magnification.

For western blot analysis, cell pellets were lysed as described previously (16 (link)). Primary antibodies were anti-phospho-p42/44 MAPK (Thr202/Tyr204) (1:1000 dilution; Cell Signaling), anti-α-tubulin (1:5000 dilution; Sigma-Aldrich), anti-β-actin (1:5000 dilution; Sigma-Aldrich), anti-fibronectin (1:500 dilution; BD Transduction), anti-vimentin (1:1000 dilution; Cell Signaling), anti-E-cadherin (1:1000 dilution; BD Transduction), anti-beta-catenin (1:1000 dilution; Cell Signaling), anti-Twist (1:1000 dilution; Santa Cruz) and anti-Slug (1:1000 dilution; Santa Cruz). Secondary antibodies were horseradish peroxidase-conjugated IgG (1:10,000 dilution; Invitrogen) for chemiluminescent signal detection and the corresponding Alexa Fluor-conjugated IgG (1:5000 dilution; Invitrogen) for fluorescence signal detection.