4.5. Immunohistochemistry and Analysis

Immunohistochemistry for phosphor-NFκB (1:100, Bethyl Laboratories, Inc, Montgomery, TX, USA) was performed on primary tumors, as described previously [16]. Paraffin-embedded tissue blocks of PCa and benign prostate tissues were processed to 4-micrometer paraffin tissue sections. Three representative tissue blocks and six sequential tissue sections per tissue block were processed for each mouse. After rehydration and serum blocking, the paraffin sections were sequentially incubated with a rabbit anti-human phosphor-NFκB polyclonal antibody with a dilution of 1:100 (Bethyl Laboratories, Inc, Montgomery, TX, USA) or anti-human Ki67 monoclonal antibody (1:100, rabbit monoclonal antibody, clone 30-9, Ventana Medical Systems, Inc., Tucson, AZ, USA) HRP-conjugated ABC kit (VECTASTAIN ABC HRP kit, VECTOR Laboratories, Burlingame, CA, USA) and followed using DAB as detection reagent (VECTASTAIN DAB kit, VECTOR Laboratories, Burlingame, CA, USA) by using an automated immunohistochemistry autostainer (Ventana BenchMark Special Stains system, Ventana Medical System, AZ, USA). Negative controls were utilized without use of primary antibody. Normal human breast cancer tissues were used as positive controls. Immunohistochemistry staining studies were repeated three times.

The H&E slides and phosphor-NFκB immunostained with counterstain slides were reviewed by PCa-specialized pathologist and regionally matched to identify the adenocarcinoma areas and the normal and benign gland areas. The immunostained slides were scanned into digital images using Aperio Digital Pathology Slide Scanner. The intensity of immunostaining of phospho-NFκB in nuclei in PCa tissue sections was determined by using Leica Aperio Nuclear Algorithm (Leica Biosystems Inc, Buffalo Grove, IL, USA) to detect the average intensity of the entire tumor section. The percentage of Ki67-positive tumor cells was calculated also by using Leica Aperio Nuclear Algorithm. The Leica Aperio Nuclear Algorithm was developed by Leica Biosystems and has been validated for clinical utilization to analyze nuclear staining biomarkers such as estrogen receptor and Ki67 in cancer tissues.