Anti ki67 antibody

HASMCs or human EA.hy926 ECs were seeded onto 12-well plates (1×10⁵ cells/1 ml/well) and incubated at 37°C in 5% CO₂ for 24 h in the same conditioning medium, followed by 48 h-incubation with the indicated concentrations of Ucn1. Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) and stained with rabbit polyclonal anti-Ki-67 antibody (Leica Biosystems, Newcastle upon Tyne, UK), followed by anti-rabbit Alexa Fluor 488 (Life Technologies, Carlsbad, CA). Terminal deoxynucleotidyl transferase- mediated deoxyuridine triphosphate-biotin nick end labelling (TUNEL) staining was performed using an In Situ Apoptosis Detection Kit (Takara Bio, Otsu, Japan). Nuclei were visualized by 4′,6-diamidino-2-phenylindole (DAPI) staining. All samples were mounted with Fluorescent Mounting Medium (Dako, Glostrup, Denmark). Fluorescence-stained cells were examined on confocal microscope (FV1000D, Olympus, Tokyo, Japan). Fluorescence was detected with wavelengths for excitation at 488 nm (Alexa Fluor 488) and 360 nm (DAPI).

Three 2.0 mm diameter core biopsies were selected from the paraffin-embedded tissue blocks. The cores were transferred to tissue microarray (TMA) and the pathologists were blinded to the identity of the TMA slides as previously described [19 (link)]. IHC tests with a rabbit monoclonal anti-Ki-67 antibody (1:1000, LEICA), an anti-MDM2 antibody (1:300, Abcam), and an anti-p53 (1:400, Abcam) antibody were performed using the Leica Bond-III system (Aperio, CA, USA). The expression of slides was examined by an Aperio AT2 digital scanner (Leica Biosystems). The H-score was obtained by multiplying the staining intensity by a constant to adjust the mean to the strongest intensity [H-score = 3 × (percentage of strong staining)] (1.0%, weak; 2.0%, moderate; 3.0%, strong) to give a score ranging from 0 to 300.

A tissue microarray (TMA) containing normal ovarian tissue (n = 40) and ovarian cancer cores (n = 40) was obtained from US Biomax (OV801). Other TMAs were developed in Imperial College as detailed in Results. Paraffin was removed with Histoclear and sections were rehydrated in graded alcohols and heated in a microwave oven at 900W for 15 min in citrate buffer pH 6. Tissue sections were pretreated using 0.3% H₂O₂ in phosphate bufferedsaline (PBS), rinsed in PBS and then incubated with 20 μl/ml normal goat serum. Primary antibodies, anti-LARP1 (SDIX-Novus Biologicals) or anti-Ki67 antibody (Leica) were diluted in PBS and incubated overnight at 4°C. Secondary or biotinylated-secondary antibodies were incubated for 30 min at room temperature and then processed with the Polymer-HRP Kit (BioGenex), or stained using the Vectastain Elite ABC Kit (Vector labs) and ImmPACT DAB (Vector labs). Tissues were then counterstained with haematoxylin. Intensity of staining was defined for each specimen (0–3, with 3 being most intense) multiplied by the percentage of cells stain-positive (to give a total score out of 300). All images were captured using a Nikon Eclipse ME600. Xenograft samples were processed in the same manner as clinical TMAs.