Ultravision lp detection system hrp polymer kit

Ki67 immunostaining was performed on 3 µm thick sections of paraffin-embedded first trimester placental tissue as previously described [6 (link)]. Briefly, after deparaffinization and antigen retrieval in 10 mM citrate buffer (pH 6) at 110 °C for 10 min, Ki67 immunohistochemistry was performed using the UltraVision LP Detection System (HRP polymer kit, Thermo Fisher Scientific, Rockford, IL, USA) as per manufacturer’s guidelines. Endogenous peroxidase was blocked with UltraVision Hydrogen Peroxide Block for 10 min at room temperature followed by a 5-min incubation with UltraVision Protein. Anti-Ki67 antibody (1:100, BD-Biosciences, Bedford, MA, USA) was diluted in Antibody Diluent (Dako, Glostrup, Denmark) and incubated for 1 h at room temperature in a humidified chamber. Antibody detection was conducted with Primary Antibody Enhancer using an HRP-labeled polymer (Dako, Glostrup, Denmark) and AEC Single Solution (Thermo Scientific, Rockford, IL, USA). Nuclei were counterstained with Mayer’s hematoxylin (Gatt Koller, Absam, Austria), and images were acquired with a Zeiss Axio Z1 microscope (Zeiss, Oberkochen, Germany) equipped with a digital camera (Olympus X, Tokyo, Japan) using the AxioVision Software (Zeiss, Oberkochen, Germany).