Using a microtome (Leica), 8 µm thick slices were taken from formalin-fixed paraffin-embedded (FFPE) prostate tissue blocks and mounted on Tomo microscope slides (Matsunami, Bellingham, WA, USA). Slides were then stained for IGF-II peptide using an IGF-II antibody (AbCam, Cambridge, UK) at a dilution of 1:600, on a Ventana BenchMark ULTRA™ machine (Roche, Oro Valley, AZ, USA). Slides were scored by two pathologists using a modified version of the Allred system, combining the proportion of tissue stained (a scale of 1–5) with the staining intensity (a scale of 1–3) to give a score out of 8 [55 ]. Currently, there is no standardized method of scoring prostate tissue.
Ventana benchmark ultra machine
Manufactured by Roche
Sourced in United States
The Ventana BenchMark ULTRA™ is an automated slide staining instrument designed for immunohistochemistry (IHC) and in situ hybridization (ISH) applications in a clinical laboratory setting. The machine automates the staining process, including dewaxing, rehydration, antigen retrieval, antibody or probe application, and counterstaining. It is intended to streamline and standardize the staining workflow for diagnostic testing.
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4 protocols using ventana benchmark ultra machine
1
Immunohistochemical Scoring of IGF-II in Prostate Tissue
2
Prostate Tissue Immunohistochemical Staining
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Using a microtome (Leica Biosystems, Nussloch, Germany), 8 µm thick slices were taken from formalin xed para n embedded (FFPE) prostate tissue blocks and mounted on Tomo microscope slides (Matsunami, Bellingham, Washington, USA). Slides were stained for IGF-II peptide using an IGF-II antibody (AbCam, Cambridge, UK) at a dilution of 1:600, on a Ventana BenchMark ULTRA™ machine (Roche, Oro Valley, Arizona, USA). Slides were scored rst by a pathologist and second, in-house, using a modi ed version of the Allred system, combining the proportion of tissue stained (a scale of 1-5) with the stain intensity (a scale of 1-3) to give a score out of 8 [35] . Currently, there is no standardised method of scoring prostate tissue.
3
COX-2 and αSMA Immunostaining Protocol
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Formalin-fixed, paraffin-embedded tissue was sectioned (3 μm), dried at 60°C, and processed in a Ventana BenchMark Ultra machine (Ventana Medical Systems Inc. (Tucson Arizona USA). Slides were incubated with monoclonal anti-COX-2 antibodies (Thermo Fischer Scientific rabbit), Universal Alkaline Phosphatase Red Detection Kit (Ultra View 760-501) and αSMA (Dako M.0851), DAB (Ultra View 760-500). Additional immunostaining on duplicates of twenty slides was performed with monoclonal COX-2 mouse antibody Invitrogen (Camarillo, CA, USA). Slides were counterstained with haematoxylin, fixed, mounted and analyzed using an inverted light microscope (Olympus, Center Valley, PA, USA).
4
Immunohistochemical Analysis of COX-2 and α-SMA
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Formalin-fixed, paraffin-embedded tissues from pancreatectomy specimens were sectioned (3 μm), and dried at 60°C. Further processing was carried out in the Ventana BenchMark Ultra machine (Ventana Medical Systems Inc. (Tucson Arizona USA) according to the manufacturer’s recommendations. Slides were incubated with monoclonal anti-COX-2 antibodies (Thermo Fischer Scientific rabbit), Universal Alkaline Phosphatase Red Detection Kit (Ultra View 760–501) and a-SMA (Dako M.0851, DAB (Ultra View 760–500). Finally, slides were counterstained with haematoxylin, fixed, mounted and analyzed using an inverted light microscope (Olympus, Center Valley, PA, USA).
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