Immunohistochemistry and Scoring

PDK1 expression levels between OCs and BOTs were semi-quantitatively compared by immunohistochemistry (IHC) scoring. Briefly, slides were dried in the oven at 70 °C for 40 min and dehydrated in xylene and graded alcohols. Antigen retrieval was performed with 0.01 M citrate buffer at pH 6.0 at 95 °C for 20 min. After cooling to room temperature, the slides were incubated with 3% hydrogen peroxide to block endogenous peroxidase activity. Then slides were incubated with PDK1 antibody (1:100, ab110025, Abcam Inc, Cambridge, MA, USA) at 4 °C overnight followed incubating with horseradish peroxidase-linked secondary antibodies at 37 °C for 15 min and stained using diaminobenzidine solution and counterstained with hematoxylin. Two independent pathologists with no prior knowledge of clinicopathologic information evaluated the IHC results.

IHC values for PDK1 were calculated by multiplying immunostaining intensity by the proportion of positive cells. The intensity was scored on a 4-point scale for cytoplasmic staining: 0 = no reactivity; 1+ = faint or weak reactivity; 2+ = moderate reactivity and 3+ = strong reactivity. The proportion of positive cells over the total number of cells was semiquantitatively evaluated and classified into 5 scores, as follows: score of 0: 0%, score of 1: 1-25%, score of 2: 26-50%, score of 3: 51-75%, and score of 4: >75%. The final score was obtained by multiplying the intensity score to proportional scores and was classified as follows: 0-2 as negative and 3-12 as positive.