Ab33766

Paraffin blocks that contained sufficient formalin-fixed tumor specimens were serial sectioned at 3 μm and mounted on silane-coated slides for immunohistochemical staining analysis. Dimethylbenzene rehydrated through 100% ethanol, 100% ethanol, 95% ethanol, 85% ethanol and 75% ethanol were applied to deparaffinize. In all, 0.01 mol/L of sodium citrate buffer (pH 6.0) was used to the progress of antigen retrieval treatment (autoclaved at 121°C, 2 min). Then, 3% H₂O₂ was applied to block endogenous peroxidase at room temperature for 10 min. The sections were washed in PBS solution subsequently and blocked with 10% goat serum (ZhongShan Biotechnology, Beijing, China) for 30 min and incubated with anti-eIF4E (ab33766, 1:100 dilution, monoclonal; Abcam, Cambridge, MA, USA) or anti-eIF4E-BP1 (ab32024, 1:100 dilution, monoclonal; Abcam) antibody or anti-vascular endothelial growth factor C (VEGFC; ab83905, 1:100 dilution, polyclonal; Abcam) or anti-phospho-4E-BP1 (Thr37/46) (236B4) (2855, 1:200 dilution, monoclonal; Cell Signaling Technology, Boston, MA, USA) at 4°C for 12 h. The sections were washed in PBS solution three times and incubated with HRP-conjugated secondary antibody for 30 min at room temperature. All slides were counterstained with diaminobenzidine (DAB) solution and 20% hematoxylin and dehydrated. The primary antibody diluent was regarded as negative control.