Anti egf

After fixation of gastric tissues in 10% formalin saline overnight, the tissues were embedded in paraffin bee wax at 56 °C for 6 h. Paraffin wax tissues were cut by rotary microtome at 5 µm thickness. Then the sections were deparaffinized and were washed with tap water and dehydrated in serial dilutions of ethyl alcohol. Then the specimens were cleared in xylene and placed in 0.3% hydrogen peroxide/methanol for 20 min to block endogenous hydrogen peroxidase activity followed by washing with PBS. After that, 10% normal goat serum was added followed by incubation overnight at 4 °C in a humid chamber with mouse monoclonal anti- PCNA (1:75) (Santa Cruz Biotechnology), rabbit polyclonal anti-TGF-β1 (1:500) (Abcam, USA), and anti-EGF (1:100) (Abcam, USA). After that, they were incubated with the biotinylated goat anti-rabbit secondary antibody (1:200, Vector Labs, Peterborough, UK) for 30 min at room temperature followed by the addition of 3,3′-diaminobenzidine (DAB) chromogen. Eventually, sections were counterstained with Mayer’s hematoxylin and examined by a light microscope. For negative control; sections were routinely processed but the primary antibodies were replaced by PBS.

Harvested cells were lysed in buffer containing 4% SDS and 2 M urea in phosphate-buffered saline (PBS). Western blotting and dot blotting were performed as we previously described^{48 (link)}. Antibodies were purchased as indicated: anti-EGFR (Abcam, Cambridge, MA), anti-GAPDH (ICN Biomedicals, Irvine, CA), anti-phospho-ERK (Santa Cruz Biotechnology, Dallas, TX), anti-14-3-3ξ (Santa Cruz Biotechnology, Dallas, TX), anti-EGF (Abcam, Cambridge, MA), anti-BAX (Cell Signaling Technology, Danvers, MA), anti-p53 (Santa Cruz Technology, Dallas, TX), anti-γH2AX (Cell Signaling Technology, Danvers, MA). HRP-conjugated species-specific secondary antibodies (KPL, Gaithersburg, MD) were visualized under LAS-4000 chemiluminescence detection system (FUJIFILM, Tokyo, Japan). Acquired images were analyzed using Image Gauge (FUJIFILM, Tokyo, Japan) according to the manufacturer’s instruction.

The protein levels were quantified by western blotting analysis of cell extracts using antibodies below: anti-GAPDH (1:2000, Santa Cruz, USA), anti-PN-1, anti-E-cadherin, anti-Vimentin, anti-Snail, anti-ZEB-1, anti-MMP9, anti-Oct4, anti-Sox2, anti-Nanog, anti-EGFR, anti-ERK1/2, anti-P-ERK1/2, anti-P-PKCδ, anti-PKCδ, anti-EGR1, anti-htrA1, and anti-EGF (1:1000, Abcam, USA). ImageJ software was used for protein bands analysis. Immunofluorescence assay was performed as described elsewhere^{15 (link)}, in which the monoclonal antibodies, including anti- PKCδ (1:100, Abcam) and anti-Vimentin (1:200, Abcam) antibodies were used. Cell images were captured using a laser scanning confocal microscopy (Olympus, Japan).