Ncl l ki67 mm1

Explant cultures were embedded in paraffin and cut into 5 μM sections, after which hematoxylin and eosin (HE) staining was performed using standard methods. For immunohistochemistry, the sections were deparaffinized, blocked with 3% hydrogen peroxide and 10 mM citrate buffer, incubated with antibodies directed against Ki67 (Novocastra, NCL-L-Ki67-MM1) or cleaved caspase 3 (Cell Signaling Technologies, clone 5A1E, #9664) at 4 °C o/n, and with a biotinylated secondary antibody for 1 h at room temperature. Next, a chromogenic HRP-mediated diaminobenzidine staining method was used (Vectastain Elite ABC Kit, Vector Laboratories, USA), after which the samples were counterstained with Mayer’s hematoxylin. The stained sections were scanned using a digital slide scanner (Pannoramic 250 Slide Scanner, 3DHistech). Five representative images from cancerous areas were taken at 40x magnification. The number of stained cells/nuclei was quantified using thresholding within the Image J program, and the total number of tumor cells was calculated manually.