Basic dab detection kit

The immunohistochemical study was approved by the local Institutional Review Board (approval number 201601370B0 and 201701722B0). Immunohistochemistry (IHC) was performed as previously described in detail [26 (link)]. In brief, 4-μm-thick formalin-fixed, paraffin-embedded tissue slices were deparaffinized in xylene and rehydrated through graded washes of ethanol in water. Sections were stained with the anti-phospho-NPM/B23 (T199) or ERα antibody using a Basic DAB detection kit (Ventana Medical Systems, Tucson, AZ, USA) in an automated IHC stainer. Hematoxylin was used for counterstaining. A semiquantitative immunostaining score (histoscore) was calculated as the percentage of positive cells multiplied by their staining intensity (0 = negative, 1 = weak, 2 = moderate, 3 = strong). Consequently, the histoscore ranged from a minimum of 0 to a maximum of 300 (i.e., 100% multiplied by 3). IHC was also performed on a commercially available endometrial cancer tissue array (EMC1021; US Biomax Inc., Rockville, MD, USA).

Immunohistochemistry was performed on one tumor tissue block, representative for the whole lesion. At first 4 µm thick paraffin sections were cut and dried overnight. For immunohistochemical analysis the following antibodies were used: CK7 (Dako, Denmark, monoclonal antibody, clone OV-TL 12/30, dilution 1/6000) and a CK20 (Dako, Denmark, monoclonal antibody, clone Ks20.8, dilution 1/400), CK8 (Becton, Dickinson and Company, monoclonal antibody, clone CAM5.2, dilution 1/400), CK19 (Dako, Denmark, monoclonal antibody, dilution 1/200) and CEA (Dako, Denmark, polyclonal, dilution 1/40.000). Tumor tissue was considered as iCCA, if CK 7 and CK 19 showed positive staining and cytokeratin 20 showed a negative staining^{65 (link)}. The stainings were carried out using an Avidin–Biotin method with antigen retrieval. The staining was performed on an automated Ventana IHC instrument with a Ventana basic DAB detection kit. Our immunohistochemical analysis was validated through positive and negative controls (by omitting the primary antibody).