Immunohistochemistry was performed on consecutive 4-µm-thick sections of formalin fixed and paraffin embedded FTSEC mouse tumors. Antigen retrieval and staining for gamma H2AX (clone and manufacturer?) was performed as previously described in ref. 57 (link). Staining for PAX8 (Roche, clone MRZ-50) and Ki-67 (Roche, clone 30-9) was performed on an automated BenchMark ULTRA staining module (Ventana) using ULTRA CC1 cell conditioning for antigen retrieval and the OptiView DAB Detection Kit (Ventana). All slides were analyzed in a blinded fashion by a gynecological pathologist.
Benchmark ultra staining module
Manufactured by Roche
Sourced in United States
The Benchmark Ultra staining module is a piece of laboratory equipment designed for automated staining of tissue samples. It provides a standardized and consistent staining process to support histological analysis.
Automatically generated - may contain errors
Lab products found in correlation
Optiview dab detection kit, by Roche (1 mentions)
Optiview detection kit, by Roche (1 mentions)
Optiview amplification kit, by Roche (1 mentions)
Ultraview dab detection kit, by Roche (1 mentions)
Hematoxylin 2, by Roche (1 mentions)
Bluing reagent, by Roche (1 mentions)
Ultraview universal dab detection kit, by Roche (1 mentions)
Ultra cc1, by Roche (1 mentions)
4 protocols using benchmark ultra staining module
1
Immunohistochemical Analysis of FTSEC Tumors
2
ALK and ROS1 Immunostaining Protocol
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For the immunohistochemical studies, 4μm wide sections were prepared from FFPE blocks and positive control was added at the edge of the slides. IHC staining was performed using ALK (clone D5F3, V790-4794, Ventana Medical Systems Inc., Oro Valley, AZ, Oro Valley, AZ, USA) and ROS1 (1:50; 3287S, Cell Signaling, Danvers, MA, USA) antibodies with the OptiView Amplification Kit (Ventana Medical Systems Inc.) in conjunction with the OptiView detection kit (Ventana Medical Systems Inc.) on a Benchmark Ultra staining module (Ventana Medical Systems Inc.). All cases were tested by a single and dedicated thoracic pathologist. As indicated by the International Association for the Study of Lung Cancer (IASLC) intensity scoring was as follows: strong staining (3+) is clearly visible using 2× or 4× objective lens, moderate staining (2+) requires a 10× or 20× objective lens and weak (1+) cannot be seen until a 40× objective is used. Cases with a diffuse staining and (3+) score were interpreted as positive.
3
Immunohistochemical Staining of PLA2R in PMN
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Renal biopsies from patients with PMN were stained for the PLA2R antigen. Formalin-fixed, paraffin-embedded (FFPE) blocks were sectioned at 3.5 µm and a positive control was added on the right edge of the slides. The slides were warmed up to 60 °C for 1 h and were processed by a fully automated protocol on a Benchmark Ultra staining module according to the manufacturer’s recommendations (Ventana medical Systems, Tucson, AZ, USA). Briefly, after sections were dewaxed and rehydrated, a Mild CC1 Benchmark Ultra pretreatment for antigen retrieval (Ventana Medical Systems Inc., USA) was selected for PLA2R (Diluted 1:1000, Sigma-Aldrich, HPA012657, St Louis, USA). Anti-PLA2R antibodies were detected with UltraView DAB Detection Kit (Ventana medical Systems, Tucson, AZ, USA). Sections were incubated for 40 min with anti-PLA2R antibodies and counterstained with Hematoxylin II (Ventana medical Systems, Tucson, AZ, USA). After the run on the automated stainer was completed, the slides were dehydrated in graded ethanols (70%, 96%, and 100%). Before cover-slipping, sections were cleared in Xylene and mounted with Entellan.
4
Automated Immunohistochemical Detection of HCMV
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All samples were subjected to H&E staining to detect signs of cytopathic damage (i.e., inclusion bodies). IHC staining for HCMV was performed on all formalin-fixed paraffin-embedded (FFPE) samples, sectioned at 4 μm on polarized slides. HCMV IHC was performed using a Benchmark ULTRA staining module (Ventana Medical Systems, Roche Diagnostics, MB, Italy) equipped with a fully automated protocol. Briefly, slides—previously baked overnight at 37 °C—were deparaffinized and rehydrated. Antigen retrieval was achieved using UltraCC1 (Ventana Medical Systems) for 36 min. The prediluted HCMV antibody (anti-HCMV blend cocktail 8B1.2, 1G5.2 & 2D4.2 mouse monoclonal primary antibody; catalogue No. 760-4703; Ventana Medical Systems) was incubated for 32 min at RT. Signal amplification was performed with the Amplification kit (Ventana Medical Systems), and detection was carried out with UltraView Universal DAB Detection kit (Ventana Medical Systems). To enhance visualization, counterstaining was performed with hematoxylin for 8 min, followed by 4-min incubation with Bluing Reagent (Ventana Medical Systems). Upon completion of the automated staining, the slides were subjected to dehydration and coverslipping through an HE600 platform (Ventana Medical Systems; Roche Diagnostics).
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