Sections (3-µm thickness) were cut from FFPE blocks. Antibody information is shown in Table I . For YAP1, the slides were pretreated for 40 min in a steamer with pH 9 Tris-EDTA buffer, and rabbit monoclonal anti-human YAP1 (dilution, 1:500) was used. For TAZ, rabbit polyclonal anti-human TAZ (dilution, 1:50) was used. IHC studies were performed using an autoimmunostainer (Leica Bond-III; Leica Biosystems, Buffalo Grove, IL, USA). IHC staining was performed using the BOND Polymer Refine Detection system kit (catalogue no. DS9800; Leica Biosystems). Sections were incubated in 3% H2O2 solution at room temperature for 5 min to block endogenous peroxidase activity. For YAP1, sections were incubated with the primary antibody at 4°C overnight, followed by incubation with the secondary antibody at room temperature for 8 min. For TAZ, sections were incubated with the primary antibody at room temperature for 8 min followed by incubation with the secondary antibody at room temperature for 8 min.
Bond polymer refine detection system kit
Manufactured by Leica
The BOND Polymer Refine Detection system kit is a laboratory equipment product designed for immunohistochemistry (IHC) and in situ hybridization (ISH) applications. The kit provides a polymer-based detection system for the visualization of target antigens or nucleic acid sequences in tissue samples.
Automatically generated - may contain errors
2 protocols using bond polymer refine detection system kit
1
Immunohistochemical Analysis of YAP1 and TAZ
2
Immunohistochemical Analysis of NRF2 and NQO1
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Immunohistochemistry was performed on a Leica Bond-III Autostainer, using the Bond Polymer Refine Detection System Kit (Leica Biosystem), according to the manufacturer’s instructions. Epitope retrieval was performed in ER2 buffer (EDTA based buffer and surfactant, pH = 9) for 30 min at 95°C for NRF2 staining and in ER1 buffer (EDTA based buffer and surfactant, pH = 6) for 20 min for NQO1 staining. Primary NRF2 antibody (Santa-Cruz Biotechnology A-10, sc-365949) was applied at 1:100 dilution for 1 h at room temperature. Primary NQO1 antibody (SIGMA-ALDRICH, HPA007308) was applied at 1:150 dilution for 20 min at room temperature. Slides were counterstained in hematoxylin and mounted in Pertex mounting medium (CellPath). Staining was categorized using a standard pathological system, with the staining intensity (+ to +++) and the percentage of tumor stained cells (0 to 100%). Samples were considered NRF2-high if nuclear staining intensity was +++ in more than 50% of tumor cells, and intermediate (+++/<50% and ++/>50%), low (++/<50% and +/>50%), or negative (+/<50% and 0).
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