Optiview dab

Tissue microarrays were stained for VHL using an Autostainer (BenchMark Ultra, Ventana Medical System, Tucson, AZ, USA). Sections were blocked for endogenous peroxidase activity using H₂O₂ and treated enzymatically with protease 1 for 4 min (760-2018, Ventana Medical System, Tucson, AZ, USA) for epitope retrieval. The TMAs were then incubated for 32 min at 36°C with the primary antibody against VHL (SC-5575, Santa Cruz Biotechnology, TX, USA) at a dilution of 1:400. Detection and visualization was carried out using OptiView-DAB (Ventana Medical System, Tucson, AZ, USA). Sections were finally counterstained with hematoxylin (Ventana Medical System, Tucson, AZ, USA).
For analysis of expression of VHL, data were distributed into two categories: normal (> 10%) vs. altered (≤10%) according to the method described by Weber et al [49 (link)].

ROS1 IHC was performed using the D4D6 clone (dilution 1:100; Cell Signaling Technology, Danvers, MA) with an ultrasensitive detection system (OptiView DAB IHC detection and amplification) on a BenchMark XT automated immunostainer (Ventana Medical Systems, Illkirch Graffenstaden, France).

Prostate biopsies and RARP specimens were fixed in formalin and embedded in paraffin. From paraffin blocks, 3-μm-thick sections were cut and the slides were then stained with hematoxylin and eosin (H&E). Only the TB-positive cores and the dominant tumor lesion of the RP specimen were further analyzed with immunohistochemistry (IHC) tests. PSMA IHC was conducted with an automatic immunohistochemistry stainer instrument, as previously described (12 (link)) (Benchmark Ultra; Ventana/Roche Group 1910 Innovation Park Dr. Tucson, AZ 85755, USA). The antigen retrieval used was cell conditioning 1 for 16 min at 99°C and the primary antibody PSMA (clone EP192, prediluted, Roche) was incubated for 16 min at 36°C. The revelation system used was OptiView DAB (12 min linker and 12 min HRP multimer) (Ventana/Roche).
Histologic evaluation and PCa staging and grading were performed by a single dedicated genitourinary pathologist in accordance with the criteria established by the World Health Organization (WHO Classification of Tumours of the Urinary System and Male Genital Organs 2022) (21 (link)).
The PSMA IHC expression was evaluated both for the intensity and the site of expression, whereas this could have been granular/cytoplasmic or membranous expression or both.

TNBC and normal tissue microarrays BR1301 and BN0811 (Biocat) were deparaffinized and treated with 1.5% H₂O₂ in Tris-buffered saline (pH 7.5) for 10 min to block peroxidase activity. Samples were then washed in TNT buffer (0.1 m Tris, 0.15 m NaCl, 0.05% Tween-20, pH 7.5) and heated to 100 °C in T-EG buffer (10 mm Tris, 0.5 mm EGTA, pH 9.0) for 48 min. This was followed by incubation with rabbit anti-HMGA2 (D1A7; Cell Signaling) diluted in antibody diluent (S2022, DAKO Cytomation, Glostrup, Denmark) for 1 h at room temperature. Then, samples were washed with TNT, incubated with OptiView DAB (Roche), washed again and incubated with 3,3′-diaminobenzidine (DAB)+ substrate-chromogen for 10 minutes. After another wash with H₂O, tissues were counterstained with Mayers hematoxylin before mounting in AquaTex (Merck Inc., Whitehouse Station, NJ, USA).