IHC was performed using the BenchMark Ultra (Ventana Medical Systems, Roche Diagnostics Division, Hoffmann-La Roche Ltd, Basel, Switzerland) automated immunostainer and visualization of the antibody-antigen reaction was via the indirect biotin-free method, ultraView Universal diaminobenzidine detection kit (Ventana Medical Systems, Roche Diagnostics Division, Hoffmann-La Roche Ltd, Basel, Switzerland). The slides were counterstained with haematoxylin. The following antibodies were used: hMLH1 (M1 clone, Ventana, 60 min heat pre-treatment, incubation time 80 min), hPMS2 (EPR3947 clone, Cell marque, 30 min heat pre-treatment, incubation time 40 min), hMSH2 (G219-1129 clone, Cell marque, 30 min heat pre-treatment, incubation time 60 min) and hMSH6 (clone 44, Ventana, 30 min heat pre-treatment, incubation time 20 min). Proof of validation for antibodies was present in the technical specification inserts provided by the manufacturers. Technical validation of adequacy of IHC was possible with external on-slide control composed of colonic mucosa (Bragoni et al. 2017 (link)) and/or internal control of colonic mucosa and/or tumour-associated stroma. The IHC laboratory at our institution has taken part in the UK NEQAS (United Kingdom National External Quality Assessment Service) quality assurance assessment runs (2022) for MMR testing.
Ultraview universal diaminobenzidine detection kit
Manufactured by Roche
The UltraView Universal diaminobenzidine detection kit is a laboratory equipment product designed for the detection and visualization of target proteins in tissue samples. It utilizes diaminobenzidine as the chromogenic substrate to produce a brown color reaction, enabling the identification and localization of specific proteins of interest.
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2 protocols using ultraview universal diaminobenzidine detection kit
1
Immunohistochemical Evaluation of MMR Proteins
2
Automated Immunohistochemistry for Protein Detection
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Immunohistochemical detection of different proteins was performed using the Ventana Discovery XT automated staining system (Ventana Medical Systems, Tucson, AZ). Paraffin-embedded tissue microarray blocks were sectioned into 4-mm slices, deparaffinized, and antigens were demasked in EDTA buffer (pH 8.4). Primary antibodies used were specific for ERG (clone EPR3864, ready to use; Ventana Medical Systems, Tucson, AZ), EP1 to EP4 (all polyclonal rabbit Ig; all Cayman Chemical, Ann Arbor, MI), or IL-6 (polyclonal rabbit Ig; Abcam, Cambridge, MA). Secondary staining was conducted using a biotin-free detection kit (UltraMap anti-Rb Detection Kit). Color was developed using alkaline phosphatase (Ultra-Map anti-Rb Alk Phos; Ventana Medical Systems) for IL-6 or 3-3 0 -diaminobenzidine (UltraView Universal Diaminobenzidine Detection kit; Ventana Medical Systems) for all other antigens. Finally, slides were counterstained with Hematoxylin II and Bluing Reagent (Ventana Medical Systems).
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