Dako pen

To examine the MET expression levels in the five gastric cancer cell lines and five PDX tumors, paraffin blocks were deparaffinized using xylene and an ethanol series (100%, 95%, 80%, and 70%). Next, the sections were washed in distilled water, and endogenous peroxidase blocking was performed with 0.3% hydrogen peroxidase (H₂O₂) for 10 min. After washing in distilled water, antigen retrieval was performed in a buffer containing 10 mM Tris, 1 mM EDTA, and 0.03% Tween 20 (pH 9.0) for 30 min in a pressure cooker. A Dako pen was used to draw a border line on the slide, and the sections were blocked with 4% BSA in PBS-T for 30 min to prevent nonspecific binding. After washing, the sections were incubated for 1 h at room temperature (RT) with primary antibody (1:100 dilution) against total MET (CONFIRM anti-MET rabbit monoclonal antibody; SP44 clone, #790-4430; Ventana Medical System, Inc., Tucson, AZ, USA), followed by incubation with HRP-conjugated secondary antibody (1:200) for 30 min at RT. After washing in PBS-T, the sections were developed by incubation with diaminobenzidine (DAB) solution for 7 min and then counterstained with Mayer’s Hematoxylin for 3 min. Finally, the sections were dehydrated and mounted, and then analyzed using a computer image analysis system [9 (link),18 (link)].

Cross-sections 2 μm in thickness were taken with a microtome (Leica MR 2145) from paraformaldehyde-fixed paraffin-embedded eye tissues, floated in a sterile bath, placed onto poly-L-Lysine-coated glass slides, and dried at room temperature. After overnight incubation at 60 °C, the slides were dewaxed in xylene for 30 min, rehydrated through a graded ethanol series (100%, 95%, 80%, and 70%, sequentially), washed in distilled H₂O and PBS for 10 min, treated with 2% trypsin containing 50 mM Tris buffer (pH 7.5) at 37 °C for 15 min, and then washed again with PBS. Sections were delineated with a Dako pen (Dako, Glostrup, Denmark), incubated in 3% H₂O₂ solution for 15 min to inhibit endogenous peroxidase activity, and washed with PBS. The slides were incubated with VEGF primary antibody at 57 °C followed by washing with PBS. Afterwards, a biotinylated secondary IgG antibody was applied and washed with PBS before incubating with the streptavidin-peroxidase conjugate (Histostain Plus, Invitrogen, Camarillo, CA, USA) for 30 min to visualize the immunostaining. The whole procedure was finished after counterstaining the sections with Mayer’s hematoxylin (Sigma Chemical Co., St. Louis, MO, USA). All sections were examined and photographed with an Olympus C-5050 digital camera mounted on an Olympus BX51 microscope (Olympus Corp., Tokyo, Japan).

The 6 µm-thick tissue slices were immersed in xylol and graded ethanol before staining for HDL using the avidin–biotin complex method according to a previous report [23 (link)]. In brief, tissue sections were rinsed with phosphate-buffered solution (PBS) prior to the antigen recovery step. The area of the tissue sections was marked with a Dako pen before treatment with hydrogen peroxidase-methanol to neutralize peroxidase endogenous to the tissue section. After a PBS rinse, the pancreatic tissue slices were then treated, at 4 °C, with HDL antibody (Abcam, Waltham, MA, USA, Cat# 34788, dilution: 1:100). After 24 h, the slides of the pancreatic sections were flushed thoroughly in PBS, and immersed in 2° biotinylated antibody (anti-rabbit IgG, dilution: 1:20, Abcam) at 22 °C. The sections were later douched in PBS and treated with 3,3-diamino-benzidine tetrahydrochloride (Sigma-Aldrich), dehydrated and placed in DPX (Dibutylphthalate Polystyrene Xylene) mounting fluid. Photoimages were taken with a digital camera fitted with Axio-Vision version 3.0 (Carl Zeiss, Oberkochen, Germany). The immunohistochemical staining was repeated 4 times.