Ventana ultra view universal dab detection kit

Four-µm-thick tissue sections on glass slides were deparaffinized at 60 °C for 1 hour, dewaxed at 72 °C for 3 minutes, and hydrated by washing with alcohol three times. Immunohistochemical staining was performed using two types of autostainer, Leica Bond-max autostainer (Leica Biosystems, Melbourne, Australia) and Ventana BenchMark XT staining system (Ventana Medical Systems, Inc., Tucson, AZ, USA). Antigens were retrieved by heat treatment in the epitope retrieval solution for each autostainer (pH 6.0 Bond epitope retrieval solution 1, pH 9.0 Bond epitope retrieval solution 2, or Ventana mild 32 condition reagent). After peroxidase blocking for 5 minutes, the primary antibody was incubated for 15 minutes. The type, dilution level, and company information of the 25 primary antibodies used are listed in Table 5. After the primary antibody reaction, a chromogenic procedure was done using the Bond polymer detection kit (Leica Biosystems, Cat. No. DS9800) or Ventana ultraView universal DAB detection kit (Ventana Medical Systems, Cat. No. 760-500). Counterstaining for nuclei was done by incubation with hematoxylin for 1 minute.

Archival formalin-fixed paraffin-embedded tumor tissues were recut for a routine hematoxylin and eosin (H&E) stain and the immunohistochemical procedure. Histological characteristics, including the cytological details of the tumor, the presence of necrosis and ulceration, angiocentricity and angiodestruction, admixed inflammatory infiltrates, and the depth of the neoplastic infiltration, were reviewed and assessed by three of the authors (BHY, XYZ and XQL) under a multi-headed microscope.
The immunohistochemical study was performed using a Ventana Bench Mark ultra autostainer (Ventana Medical System Inc., Roche Tuson, AZ, USA) and the Ventana ultra view universal DAB detection kit. The primary antibodies against CD20, CD2, cytoplasmic CD3 [CD3 (epsilon)], CD4, CD5, CD7, CD8, CD30, CD56, T-cell-restricted intracellular antigen-1 (TIA-1), granzyme B (GrB), perforin, ALK1 and Ki-67 were employed in the present study. Except for TIA-1 (Dako, Glostrup, Denmark), all of the above-mentioned antibodies were commercial products by Roche Ventana. For each stain, a parallel stain using appropriate positive and negative controls was performed. The Ki-67 labeling index was estimated to the closest decile.

Paraffin blocks were sectioned at approximately 3–5 microns thickness. Sections were placed on SuperFrost Plus™ glass slides. Slides were incubated overnight at 64 °C. Slides were stained using the standard procedure in Ventana BenchMark Ultra automated slide stainer in combination with Ventana UltraView Universal DAB Detection Kit (Ventana, Roche Diagnostics cat #760-500). The slides were stained with the following antibodies: monoclonal mouse anti-Human Ki-67, clone MIB-1 (with Nuclear Dekloaker Dako as heat mediated antigen retrieval solution, cat# M7240), diluted 1:200, mouse anti-Human E-cadherin (with Nuclear Dekloaker Dako as heat mediated antigen retrieval solution Invitrogen, cat# #33-400, diluted 1:50, mouse anti-Human CK-7(with citrate as heat mediated antigen retrieval solution, Dako, cat#7019), diluted 1:200, mouse anti-Human CK-18 (with citrate as heat mediated antigen retrieval solution, Dako, cat#7010), diluted 1:3000, mouse anti-Human GATA-3 (with citrate as heat mediated antigen retrieval solution, Zytomed, cat#BMS054), ready to use.

Cholesterol (purity 94%, Sigma-Aldrich, Japan), cholic acid (purity >98%, Sigma-Aldrich, New Zealand), and eugenol (purity 99%, Sigma-Aldrich, Germany) were purchased from Sigma-Aldrich in a high-purity grade. Atorvastatin calcium was kindly provided by SANA Pharmaceuticals, Amman, Jordan. The enzymatic kits for quantitative assay of TC, TG, HDL, AST, ALT, ALP and LDH, were purchased from Biosystem, Barcelona, Spain. Commercial assay kits used to determine the activity of catalase enzyme (CAT) (Cayman’s Catalase Assay Kit, Item No. 707002) and superoxide dismutase enzyme (SOD) (Cayman’s Superoxide Dismutase Assay Kit, Item No. 706002) were purchased from Cayman Chemical Company, Ann Arbor, U.S.A. Primary rabbit polyoclonal anti-TRPV1 antibody was bought from Novus Biologicals (Cat no. NB100-1617; Novus Biologicals, Littleton, CO 80120 USA). Ventana ultraView universal DAB detection kit (Cat no. 760-500), Ventana Liquid Coverslip LCS (Cat no. 650-010), Ventana Reaction Buffer (Cat no. 950-300), Ventana EZ Prep solution (Cat no. 950-102), Ventana cell conditioning solution (CC1) (Cat no. 950-124), Ventana Amplification kit (Cat no. 760-080), hematoxylin II (Cat no. 790-2208), Bluing reagent (Cat no. 760-2037) were purchased from Ventana Medical Systems Inc., Tucson, Arizona, USA.

Formalin-fixed paraffin-embedded tissue blocks were available for all cases and recut for routine hematoxylin and eosin (H&E) staining and the immunohistochemical procedure. Histological characteristics were examined by two experienced hematopathologists (BHY and XQL). Immunohistochemical studies were performed on these tissue sections, using a Ventana BenchMark Ultra Autostainer (Ventana Medical System Inc., Roche Tucson, AZ, USA) and the Ventana ultraView Universal DAB Detection Kit. The primary antibodies included cytoplasmic CD3, CD4, CD8, CD20, CD30, CD43, CD56, T-cell-restricted intracellular antigen-1 (TIA-1), ALK1, EMA and Ki67. All of these antibodies were commercial products from Roche Ventana. All stainings were performed with appropriate positive and negative controls.

Various IHC stains of relevance were performed in the clinical setting at the Dept. of Pathology, Lund, Region Skåne, Sweden, as part of the histopathological diagnostic procedure. Four micrometer thick sections from FFPE tissue blocks were pre-treated and stained in a Ventana Bench-Mark Ultra using Ventana ultraView Universal DAB Detection Kit (Ventana Medical Systems, Tucson, AZ). Recommended control tissue was used on each slide. The clones and vendors for the antibodies included in this study were TTF-1 clone 8G7G3/1, CK7 clone OV-TL 12/30, CK20 clone SP33, CDX2 clone EPR2764Y, S100 polyclonal, estrogen receptor clone SP1, progesterone receptor clone 1E2, HER2 clone 4B5, ALK clone D5F3, all Ventana Medical Systems (Tucson, AZ), napsin A clone IP64, CK5 clone XM26, both Novocastra/Leica Biosystems (Kista, Sweden), p63 clone 4A4, smooth muscle specific actin clone 1A4, both Dako (Glostrup, Denmark), p40 clone BC28, Histolab Products/Biocare Medical (Gothenburg, Sweden), and GATA3 clone L50-823, Cell Marque (Rocklin, CA).

Commercially available enzyme immune assay (Complement System Screen Wieslab; Euro Diagnostica, Malmö, Sweden) and murine anti-human C5b-9 antibody (clone aE11, Dako, Glostrup, Denmark) were used according to manufacturer’s instructions to detect functional complement activity and sC5b-9 production in plasma, respectively. Both methods detect the respective human and pig epitopes [41 (link)]. In tissue, the membrane form of C5b-9 was visualized in frozen sections from the AAR, border zone and control area. Tissue samples were incubated for 30 min at room temperature using the murine anti-human C5b-9 antibody (clone aE11, Dako, Glostrup, Denmark) diluted 1/25 in Dako antibody diluent (Dako K8006, Glostrup, Denmark), washed in phosphate buffered saline and stained by Ventana ultra View Universal DAB Detection Kit (Ventana Medical Systems, Inc., Tucson, AZ) according to the manufacturer’s instructions. A Nikon Eclipse E1000M microscope was used and photos were obtained with original 40× magnification.