Anti ki67 monoclonal antibody

Tissues for IHC were fixed in 10% buffered formalin for 24 h and embedded in paraffin. The deparaffinized and rehydrated sections were blocked for endogenous peroxidase by incubation in 3% hydrogen peroxide followed with antigen retrieval in the boiling citrate buffer (10 mM, pH 6.0) for 10 min. The sections were then blocked with normal goat serum (1:10) and subject to incubation with anti-Ki67 monoclonal antibody (1:100, Dako, Glostrup, Denmark) or anti-Caspase 3 antibody (1:100, Abcam, #ab32351) overnight at 4 °C. Thereafter, the PBS cleaned sides were incubated with the biotinylated secondary antibody at 37 °C for 30 min, and subsequently incubated with a 1:200 streptavidin-biotin-peroxidase complex (Sigma, St. Louis). Reactive products were visualized with 3,3′-diaminobenzidene (DAB) as the chromogen, and nuclei were counter-stained with hematoxylin.

Triplicate cores were made of endometrial tumors pre- (endometrial biopsy) and postmetformin treatment (hysterectomy), and tissue microarrays were created. Immunohistochemical analysis was performed on 4-μmol/L sections of formalin-fixed, paraffin-embedded tissues using standard methodologies. The primary antibodies included the following: (1) anti-Ki-67 monoclonal antibody, M7240, Dako (Carpinteria, CA), (2) antiestrogen receptor (ER) monoclonal antibody, 249R-16, Cell Marque (Rocklin, CA), (3) antiprogesterone receptor (PR) monoclonal antibody, 323R-16, Cell Marque (4) antiphosphorylated AMPKα monoclonal antibody, 2535, Cell Signaling Technology (Danvers, MA), (5) antiphosphorylated Akt (ser 473) monoclonal antibody, 4060, Cell Signaling Technology, (6) antiphosphorylated S6 ribosomal protein monoclonal antibody, 4858, Cell Signaling Technology, and (7) antiphosphorylated 4E-BP-1 monoclonal antibody, 2855, Cell Signaling Technology. Negative controls (lacking primary antibody) were performed for each antibody. Individual slides were scanned using the Aperio™ ScanScope (Aperio Technologies, Vista, CA), and digital images were analyzed using Aperio™ ImageScope software. This work was performed with the assistance of the UNC-CH Translational Pathology Laboratory (TPL) Core.

Tumor tissues were fixed in 10% buffered formalin for 24 h and embedded in paraffin. The deparaffinized and rehydrated sections were blocked for endogenous peroxidase by 20 min incubation in 3% hydrogen peroxide followed with antigen retrieval at 121^°C in citrate buffer (10 mM, pH6.0) for 10 min. After free cooling to room temperature, the sections were blocked for non-specific binding with normal goat serum (1:10) for 30 min at room temperature, and subjected to incubation with anti-Ki67 monoclonal antibody (1:100, Dako, Glostrup, Denmark) overnight at 4^°C. The next day, the sides were washed and incubated with the biotinylated secondary antibody at 37^°C for 30 min, and subsequently incubated with a 1:200 diluted streptavidin-biotin-peroxidase complex (Sigma, St. Louis) for 30 min. Reactive products were visualized with 3,3’-diaminobenzidene (DAB) as the chromogen, and nuclei were counter-stained with hematoxylin.