*2.10. Histopathology and Immunohistochemistry*

For histopathological analysis, tumors were fixed in 10% neutral bu ffered formalin and embedded in para ffin. General tissue morphology was visualized by standard hematoxylin and eosin staining. Next, immunohistochemical staining was performed with an ImmPRESS Reagent Kit (Vector Laboratories, Inc., Burlingame, CA, USA). Antigen retrieval was performed using 10 mM sodium citrate bu ffer (pH 6.0) for AR, NSE, Ki-67, and CD31. Antigen Unmasking Solution (Vector Laboratories, Inc.) was used for PSA. The antigen-antibody reaction was visualized using 3-,3-diaminobenzidine tertahydrochloride as a substrate. Sections were counterstained with hematoxylin and examined by light microscopy.

Cell proliferation in tumors was determined by the percentage of Ki67-positive nuclei in 10 di fferent areas at 400× magnification from each tissue specimen. A 'microvessel' was defined as mouse-specific CD31-positive endothelial cells that formed a vascular lumen. The number of microvessels was counted in 10 di fferent areas at 400× magnification from each tissue specimen, as previously described [27]. The results were independently reviewed by 2 blinded investigators.
