Surgical specimens were routinely formalin-fixed, paraffin-embedded and sectioned at 4 µm thickness. Immunohistochemical staining was performed on a BenchMark ULTRA IHC Staining module using OptiView DAB/Ventana, Roche, according to OptiView DAB IHC v5 protocol. Sections were first deparaffinized. Antigen retrieval was achieved by incubating the sections at 100 °C, pH 8.5 for 32 min. The sections were incubated in hydrogen peroxide for peroxidase inhibition, followed by incubation with the primary antibody. The antibodies used are listed in Supplementary Table 13 .
Optiview dab ihc
Manufactured by Roche
The OptiView DAB IHC is a laboratory equipment product from Roche. It is designed for chromogenic immunohistochemistry (IHC) staining, specifically utilizing 3,3'-Diaminobenzidine (DAB) as the chromogen. The product enables the visualization of target proteins in biological samples.
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Immunohistochemical Staining Protocol
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Immunohistochemical Detection of Trk Proteins
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Immunohistochemistry for Trk was performed on 4 micron thick sections. The primary antibody was a rabbit monoclonal anti-Trk (pan) covering Trk A, B and C (clone A7H6R, 92991 S, Ozyme, Cell Signaling). A dilution of 1/50 was used with 52 min incubation on a Benchmark ULTRA (Roche-Ventana, Tucson, AZ) and detection Kit Optiview DAB IHC (reference: 760-500). Heat-induced antigen retrieval was performed using Cell Ventana Conditioning buffer (CC1 standard) for 64 min. An infantile fibrosarcoma with a known NTRK fusion served as a positive external control.
A variety of other immunohistochemical stains were also undertaken. Most of these were performed at the various institutions during the original reporting of the neoplasms while others were undertaken during the preparation of this manuscript.
A variety of other immunohistochemical stains were also undertaken. Most of these were performed at the various institutions during the original reporting of the neoplasms while others were undertaken during the preparation of this manuscript.
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