Immunohistochemistry

Surgically excised tumor specimens and xenograft tumour were fixed in 4% neutral formalin at room temperature for 24 h, embedded in paraffin and cut into 4 µm sections. The sections were then deparaffinized in xylene, rehydrated in a graded alcohol series and treated with boiling 0.01 mol/l citrate buffer 15 min for antigen retrieval. Endogenous peroxidase activity was blocked with hydrogen peroxide (0.3%) at room temperature for 15 min, and the sections were incubated with 5% bovine serum albumin (R&D Systems, Minneapolis, MN, USA) at 37°C for 45 min to reduce non-specific binding. Immunostaining with PLK4 rabbit polyclonal antibodies (Table I) was carried out at 4°C for 16 h, followed by incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody from the Envision kit (Dako; Agilent Technologies, Inc., Santa Clara, CA, USA; Table I) for 45 min at room temperature. Antibody binding was detected by DAB (Dako; Agilent Technologies, Inc.), according to manufacturer’s protocol, at room temperature for 1 min and the reaction was terminated by immersion of tissue sections in distilled water once brown staining appeared. Tissue sections were counterstained with 1% hematoxylin at room temperature for 3 min and dehydrated in a graded series of ethanol. The xenograft tumour tissues were also immunostained with PLK4, Ki-67 and β-catenin primary antibodies (Table I) using the same protocol. Immunohistochemical scores were obtained by multiplying the percentage score to the intensity score of positively stained cells; images of representative fields were obtained from Nikon Digital ECLIPSE C1 microscope (Nikon Corporation) (21). Analysis was independently performed by two certified pathologists, which were blinded to the clinical and demographic characteristics of the patients. The expression status represents the average of the independent readings. An overall score of >6 and ≤6 was defined as high and low expression, respectively.

Antibodies used in the present study.

HRP, horseradish peroxidase; IgG, immunoglobulin G; IHC, immunohistochemistry; GSK3β, glycogen synthase kinase 3β; MMP, matrix metalloproteinase; p, phosphorylated; PLK4, polo-like kinase 4; WB, western blotting.