IHC for brachyury was reviewed in all cases. IHC for SMARCB1 was performed on 4-µm-whole sections of formalin-fixed, paraffin embedded tissue blocks. The primary antibody used was a mouse monoclonal anti-BAF47 antibody (1:30; BD bioscience, San Jose, CA). Antigen retrieval, antibody incubation, and chromogen counterstaining were performed in a BenchMark ULTRA automated immunostainer (Ventana, Tucson, AZ) as previously described.10 (link)
Benchmark ultra automated immunostainer
Manufactured by Roche
Sourced in United States
The BenchMark ULTRA automated immunostainer is a diagnostic instrument designed for immunohistochemistry (IHC) and in situ hybridization (ISH) applications. It automates the staining process, providing consistent and reproducible results.
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Lab products found in correlation
5 protocols using benchmark ultra automated immunostainer
1
Immunohistochemical Analysis of Brachyury and SMARCB1
2
DLBCL Tumor Classification and Subtyping
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Classification and subtyping of all tumors followed the definitions of the 2008 WHO classification of DLBCL [19] . Immunostainings for CD3, CD5, CD20,CD79a, CD10, MUM1, BCL2, BCL6, kappa and lambda light chains and heavy chain IgM (all purchased by DAKO, Denmark) were performed using Dako automated immunostainer (DAKO, Denmark). Immunohistochemistry with anti-MYC (clone Y69, Ventana-Roche, Italy) was conducted using BenchMark Ultra automated immunostainer (Ventana Medical Systems, Tucson, AZ, USA). The Hans algorithm [22] (link) was used in order to classify cases as GCB-type or non GCB-type. Immunostaining results for BCL2 and MYC were recorded as the percentage of positive cells in increments of 10% regardless of the intensity of the staining. Cases were considered as negative if <5% of tumor cells were positive. Immunohistochemical and morphological analyses were independently evaluated by two experienced hematopathologists (LR, ADN). Disagreements were resolved by joint review on a multi-head microscope.
3
Immunohistochemical Evaluation of p16 Expression
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After deparaffinisation and rehydration, heat-induced antigen retrieval was performed by microwave oven for 20 min in a citrate buffer. Endogenous peroxidase was blocked by 0.3% hydrogen peroxide in methanol for 10 min, and then nonspecific antibody binding was blocked by 10% normal goat serum. IHC was performed using the Benchmark Ultra automated immunostainer (Ventana Medical Systems, AZ, USA). Immunoreactivity against anti-p16 mouse monoclonal antibody (clone JC8, Santa Cruz Biotechnology, Inc., CA, USA) was visualised using the iVIEW detection kit (Ventana Medical Systems) containing the biotin-conjugated secondary antibody, streptavidin-hydrogen peroxide conjugate, and diaminobenzidine. The p16 IHC was interpreted as ‘positive’ when the percentage of stained nuclear and cytoplasmic cells was > 70% of cancer cells. Patchy or focal expression in the surface epithelial cells was regarded as negative.
4
HPV-associated Tumor Identification via p16 IHC
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IHC was performed using p16 as a surrogate marker to identify HPV-associated tumors. H&E (Fig. S2A andC) and IHC staining were performed on representative whole sections of tumor from formalin-fixed, paraffinembedded (FFPE) specimens, and the paraffin blocks were sectioned at a thickness of 4 μm. IHC was performed using a Ventana Benchmark Ultra automated immunostainer (Tucson, AZ, USA) and a mouse monoclonal antibody specific for p16 (E6H4, prediluted, Roche diagnostics) according to standard protocols and with appropriate positive and negative controls. A case was classified as an HPV-associated tumor only if it showed block-positive p16 staining, with diffuse nuclear or nuclear and cytoplasmic positivity (Fig. S2B). Negative results (Fig. S2D) were defined as total absence of staining or weak, focal, and discontinuous staining, which indicated HPV-independent tumors. The results of p53 immunohistochemical staining analyses were available for 16 out of 19 cases. Staining was performed using a p53 monoclonal antibody (DO7, prediluted, Agilent/Dako, Glostrup, Denmark) in all cases.
5
Immunohistochemical Characterization of Lymphoma
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