Benchmark ultra ihc ish staining system

Nuclear expressions of a-thalassemia/mental retardation X-linked syndrome (ATRX) and p53 were demonstrated immunohistochemically using a rabbit polyclonal antibody (HPA001906, 1:100, Atlas Antibodies, Sweden) and a monoclonal antibody (DO7, Ready-to-use, Ventana Medical Systems Inc), respectively. The two immunohistochemical staining protocols were performed for all tumors using the BenchMark Ultra IHC/ISH staining system (Ventana Medical Systems Inc).

Sections from patients with of DA, AA and GBM were stained with a mIDH1R132H antibody (mIDH1R132H, clone H14, Dionova, 1:100) to detect the most common isocitrate dehydrogenase (IDH) mutation using the BenchMark Ultra IHC/ISH staining system (Ventana Medical Systems, Inc, USA) as previously described [37 (link), 38 (link)].

Immunohistochemical staining was carried out using the BenchMark Ultra IHC/ISH staining system (Ventana Medical Systems, Inc, AZ, USA). The primary antibody was Ki-67/ MIB-1 (monoclonal mouse antibody, Dako, no. M7240). To detect Ki-67 labeling in non-neoplastic cells a cocktail of antibodies against CD45 (monoclonal mouse antibody, Dako, no. M0701), CD31 (monoclonal mouse antibody, Dako, no. M0823), Alpha-smooth muscle actin (ASMA) (monoclonal rabbit antibody, Spring Bioscience no. M4710) and ionized calcium-binding adaptor molecule-1 (Iba1) (polyclonal rabbit antibody, Wako) was used for double immunohistochemical staining. For Ki-67 a detection system UVDAB-HRP plus amplification followed by application of the uDAB-kit (ref. no. 760–500) was used. For the other antibodies, we used the detection system UVRed-AP followed by application of the uRed-kit (ref. no. 760–501). Nuclear counter staining was performed using Hematoxylin II (Ventana Medical Systems, Tucson, AZ) at the BenchMark Ultra instrument. Finally, slides were washed, dehydrated, and coverslipped using a Tissue-Tek Film coverslipper (Sakura, Alphen aan den Rijn, The Netherlands). The slides were scanned on the Hamamatsu whole-slide scanner (Hamamatsu, Hamamatsu City, Japan).

IHC staining was performed on the formalin-fixed and paraffin-embedded tissue specimens of cervical carcinoma in situ, collected after conizations at the National Cancer institute of Lithuania. IHC staining was performed on the fully automated BenchMark ULTRA IHC/ISH Staining system (Roche) at the National Center of Pathology (Vilnius, Lithuania), using EnVision FLEX, High pH kit (Dako, Agilent, K8023, Santa Clara, CA, USA), affinity purified MAb H7 (0.02 mg/mL) and hematoxylin (Merck, 75290, Burlington, MA, USA) as a nuclear counterstain. Unrelated in-house PPV-targeting MAb of IgG1 isotype was used as negative control. Stained tissue sections were analyzed and photographed using ScanScope XT system (Aperio, Leica Biosystems Inc., Buffalo Grove, IL, USA).