Anti ki67 clone 16a8

Immunopathological analysis was provided by the iPATH.Berlin—Immunopathology for Experimental Models, core unit of the Charité—Universitätsmedizin Berlin (Berlin, Germany). Formalin-fixed tissue samples were embedded in paraffin and 1–2 μm thick sections were cut from paraffin blocks. Sections were dewaxed and histochemically stained with H&E (hematoxylin&eosin) for evaluation of histomorphology or subjected to a heat-induced epitope retrieval step prior to incubation with anti-Ki67 (clone 16A8, BioLegend), anticleaved caspase 3 (clone 5A1E, Cell Signaling). For detection of Ki67 and cleaved caspase3, sections were incubated with secondary antibody (biotinylated rabbit antirat and biotinylated donkey antirabbit, respectively; Dianova). Biotin was detected by streptavidin coupled with alkaline phosphatase (AP) and RED as chromogen (both Dako REAL™ Detection System, Alkaline Phosphatase/RED, Rabbit/Mouse, Agilent Technologies). Nuclei were counterstained with hematoxylin (Merck Millipore) and slides coverslipped with glycerol gelatine (Merck). Whole slide scans were acquired by a Vectra® 3 imaging system (PerkinElmer).