Correlation of MR Imaging and MR Spectroscopic Imaging Findings with Ki-67, Phospho-Akt, and Androgen Receptor Expression in Prostate Cancer -- Shukla-Dave et al. 250 (3): 803 Data Supplement - Correlation of MR Imaging and MR Spectroscopic Imaging Findings with Ki-67, Phospho-Akt, and Androgen Receptor Expression in Prostate Cancer -- Radiology

Correlation of MR Imaging and MR Spectroscopic Imaging Findings with Ki-67, Phospho-Akt, and Androgen Receptor Expression in Prostate Cancer

Appendix E1

Phospho-Akt (Ser473) rabbit monoclonal antibody clone 736E11 (Cell Signaling Technology, Danvers, Mass) was used at a 1:50 dilution; AR mouse monoclonal antibody clone AR441 (DakoCytomation, Carpinteria, Calif) was used at a 1:50 dilution; Ki67 mouse monoclonal antibody clone MIB1 (DakoCytomation) was used at a 1:1000 dilution. The avidin-biotin peroxidase method was used for IHC staining.

Briefly, 6-μm whole-mount sections were deparaffinized in xylene and rehydrated in graded alcohol. Endogenous peroxidase was blocked by immersing slides in 0.1% phosphate-buffered saline/peroxidase for 15 minutes. For antigen retrieval, slides were heated in 0.01 M citric acid (pH 6) in a microwave oven for 15 minutes. After the slides were cooled to room temperature, appropriate blocking sera were applied and the slides were incubated for 30 minutes, followed by overnight incubation at 40C with primary antibodies. Optimal dilutions for each antibody had been determined in previous experiments. After extensive washing, adequate secondary antibodies were applied and the slides were incubated for 30 minutes, followed by application of avidin-biotin complex for an additional 30 minutes. Diaminobenzidine was used as the final chromogen, and the slides were then counterstained with H-E, dehydrated, and mounted.