Immunohistochemistry

The immunohistochemistry expression of the oncoprotein Her2/neu and the proliferation index Ki67 were tested on the same set of tumors. The primary antibodies used were: mouse anti-human Her2 (CB11) and mouse anti-human Ki67 (MM1) (NovoCastra, Newcastle, UK). After deparaffinization Sections were subsequently hydrated, incubated for 30 min in 1% hydrogen peroxide to block endogenous activity, and then antigen retrieval was performed by incubating the sections in a 0.01 M citrate buffer (epitope retrieval solution pH 6.0; Leica Microsystems GmbH, Wetzlar, Germany) for 30 min at 98 °C. The primary antibodies were applied for 1 h at 4 °C, with a dilution of 1:40 for Her2 and 1:200 for Ki-67. The sections were then incubated at room temperature with post primary block for 30 min to block nonspecific polymer binding. The sections were incubated with a NovoLink™ Polymer for 30 min at room temperature, followed by incubations with 3,3’-diaminobenzidine (DAB) working solution for 5 min at room temperature to develop peroxidase activity. The slides were counterstained with hematoxylin and mounted. Staining specificity was checked using negative controls. Subsequently, the primary antibodies were applied for 1 h at 4 °C, with a dilution of 1:40 for Her2 and 1:200 for Ki-67. The sections were then incubated at room temperature with post primary block for 30 min to block nonspecific polymer binding. The sections were incubated with a NovoLink™ Polymer for 30 min at room temperature, followed by incubations with 3,3’-diaminobenzidine (DAB) working solution for 5 min at room temperature to develop peroxidase activity. The slides were counterstained with hematoxylin and mounted. Staining specificity was checked using negative controls. This part has been previously described by Oueslati et al, 2017 [18].