Ultravision quanto detection system hrp polymer

Serial sections with a thickness of 4 μm were applied to the SuperFrost adhesive slides (Thermo Scientific, Waltham, MA, USA). The deparaffined sections were subjected to the antigen unmasking by the heat treatment in 0.1 M citrate buffer (pH 6.0) at of 95–98 °C. The “UltraVision Quanto Detection System HRP Polymer” (Thermo Scientific, Waltham, MA, USA) was used to visualize the IHC. Endogenous peroxidase activity was blocked by 3% hydrogen peroxide. “Ultra V Block” was used to prevent non-specific reactions and background staining. Reaction was amplified with “Primary Antibody Amplifier Quanto”. Diaminobenzidine (DAB) was used as a chromogen. The nuclei were counterstained with Mayer’s hematoxylin. Primary antibodies against osteopontin (OPN) (Abcam, ab-8448, dilution 1:300) were used. In general, the procedure of immunohistochemical staining of tissue was performed according to the previously described method [14 (link)]. At least six different fields of view (FOV) with diameter of 1 mm were analyzed for each sample. The IHC results were presented as a mean number of OPN-positive cells per FOV.

Tissue microarrays were produced from paraffin-embedded tissue blocks from 41 PDAC surgical specimens. All cases were immunostained with a sensitive non-biotin detection system (NovoLink polymer, Novocastra), with diaminobenzidine development. Heat induced antigen retrieval was performed using citrate buffer 0.01 M, pH 6.0 or Tris-EDTA pH 9.0 in a water bath for 30′. Immunostaining was performed using the anti-ASC Ab (Enzo Life Science) and Ultravision Quanto Detection System HRP polymer (Thermo Scientific). Samples were considered positive if the percentage of positive cells was superior to 10%.

Melanoma sections were stained by specific antibodies (supplementary Table S1) for ki67, cleaved Caspase 3, phospho-ERK, and COX2 after antigen retrieval performed by heating in a pressure cooker with 0,5 mM EDTA pH 8 for 15 or 20 min and using a peroxidase-labelled polymer (UltraVision Quanto Detection System HRP Polymer, Thermo Fisher Scientific) and brown DAB as a chromogen (Dako Agilent, Glostrup, Denmark). Sections were scanned using the Aperio ScanScope XT systems (Aperio Technologies, Leica Microsystems).