Biocare blocking reagent

Tumors were fixed in formalin, embedded in paraffin, cut into 4-μm thick sections, placed on glass slides (one section/slide), and processed for either hematoxylin and eosin (H&E) or immunohistochemical (IHC) staining. Slides were deparaffinized in xylene and hydrated, and antigens were retrieved by microwaving slides for 10 min with 10 mM citrate buffer. Endogenous peroxidase activity was quenched by exposing slides to 3 % hydrogen peroxide for 10 min. Following 30 min blocking with Biocare blocking reagent (Biocare Medical, Concord, CA) was followed by incubation with primary antibody diluted in the same blocking buffer as follows: Ki-67 (Dako, Carpenteria, CA; 1:200, 4 °C overnight); slides were washed with PBS, secondary antibody was applied for 30 min at RT, followed by another three washes with PBS. Diaminobenzidine (DAB) was used to develop the antibody stain followed by a hematoxylin counterstain to visualize nuclei. Slides were scanned and digitized using the Aperio Scanscope System (Scanscope XT; Aperio Technologies, Vista, CA).

Tumor tissues were fixed in formalin and embedded in paraffin using the Microm Tissue Embedding Center (Labequip, Ltd, Markham, Ontario). Then the secretions were cut (5 μm) and stained with H&E. For immunohistochemical staining, sections were hydrated and blocked with 4 % H₂O₂ in water for 15 min. Antigen retrieval was done with 20 μmol/L citrate buffer (pH 6.0) for 15 min followed by a 20-min cooldown and washed in TBS with Tween (TTBS: 50 μmol/L Tris–HCl (pH 7.5), 150 mmol/L NaCl, 0.1 % Tween 20). Slides were treated with Biocare blocking reagent (Biocare Medical) for 15 min to remove nonspecific binding. Slides were then incubated with antibodies against CPEB4 (Sigma, Shanghai) for 40 min. Then the slides were washed in TTBS for 40 min at 20 °C. After washing, the slides were incubated with antigoat horseradish peroxidase-conjugated secondary antibodies (BioGenex, San Francisco) for 40 min at 20 °C.