Sp4 r

Formalin-fi xed paraffi n-embedded breast cancer cells were examined histopathologically using haematoxylin and eosin (H&E) stain. Cell blocks were sectioned at a 4-μm thickness using a semiautomated rotary microtome (Leica RM2245, Leica Microsystems Inc., Bannockburn, IL, USA). The sections were stained with hematoxylin and eosin (H&E) with an automated side stainer and cover slipper (Tissue-Tek Prisma/Film, Sakura Finetek Inc., CA, USA). Then, the slides were examined under a light microscope (Olympus BX51, Tokyo, Japan). Formalin-fi xed paraffi n-embedded blocks were sectioned (4-μm thick) for immunohistochemical analysis. Immunohistochemical staining was performed on an automated immunohistochemistry slide staining system (Roche Ventana Bench-Mark GX, Ventana Medical Systems, Inc., Tucson, AZ, USA). Ki-67 (rabbit monoclonal primary antibody, clone 30-9, Roche), Bcl-2 (rabbit monoclonal primary antibody, clone SP66, Roche), Cyclin-D1 (rabbit monoclonal primary antibody, clone SP4-R, Roche), and BAX (rabbit polyclonal primary antibody, Dako) were analyzed on whole formalin-fi xed paraffi n-embedded cell block sections. For negative controls, the primary antibodies were omitted.