Bond enzyme pretreatment kit

For paraffin-embedded tissues, an initial automated dewaxing and rehydration step followed by heat-induced (100° C for 20 min) or enzyme-induced (10 to 15 min, Bond Enzyme Pretreatment Kit, Leica Biosystems, Wetzlar, Alemania) antigen retrieval was performed. Heat-induced antigen retrieval was performed using pH 8.8 ethylenediaminetetraacetic acid (EDTA)-based ready-to-use solution (Leica Biosystems). Slides were subsequently incubated with 3% hydrogen peroxide (5 min), optimally diluted primary antibody (15 to 30 min), a postprimary blocking reagent (to prevent nonspecific polymer binding) (8 min), horseradish peroxidase-labeled polymer (8 min), and diaminobenzidine substrate (10 min). All reagents were components of the Bond Polymer Refine detection system (Leica Biosystems). New adhesive labels needed for the second staining procedure were applied to the slides. A second immunophosphatase (AP) procedure was then performed, omitting the dewaxing, rehydration and epitope retrieval steps. The primary antibody was applied for 40 min, followed by incubation with postprimary AP blocking reagent (20 min) and AP-labeled polymer (30 min), both of which are components of the Bond Polymer AP Red detection system (Leica Biosystems). The AP reaction was carried out with the Fast Red substrate included in the Bond Polymer AP Red detection system. Hematoxylin counterstaining was performed.

Five-micrometer-thick tissue sections were cut and mounted on SuperFrost^TM Plus slides (Thermo Fisher Scientific, Waltham, MA, USA) and stored at 4 °C until the staining. Immunostaining was carried out using the Novolink Polymer Detection System (Leica-Novocastra, Nussloch, Germany) according to the manufacturer’s protocol. Slides were dewaxed using xylene and rehydrated in graded alcohol series. Endogenous peroxidases were blocked with 10%H₂O₂ in methanol (20 min, room temperature). Antigen retrieval was performed at 37 °C for 5 min using the Bond Enzyme Pretreatment Kit (Leica Biosystems). After blocking non-specific binding with the Novocastra Protein Block (10 min, room temperature), slides were incubated with mouse polyclonal anti-ZNF554 antibody (Abnova, Taipei, Taiwan) at 1:50 dilution overnight at 4 °C. After post-primary amplification (30 min, room temperature), slides were incubated with the Novolink Polymer (30 min, room temperature) and visualized with 3,3′-diaminobenzidine (DAB) for 10 min. Finally, sections were counterstained with hematoxylin, and these were mounted (DPX Mountant; Sigma-Aldrich, St. Louis, MO, USA) after dehydration. The primary antibody was omitted in negative controls.