Envision flex hematoxylin

Samples were immersion fixed in 10% neutral buffered formalin for 24 h and embedded in paraffin routinely. Sections 4 µm-thick were mounted on FLEX IHC microscope slides (Agilent, Carpinteria, CA, USA). After deparaffination and epitope retrieval (for 20 min at 97 °C in EnVision FLEX target retrieval solution at low pH for FABP4 and high pH for the remaining antibodies), immunohistochemistry was automatically performed using an AutostainerLink 48 immunostainer (Agilent). Briefly, the slides were incubated at room temperature in: (1) rabbit polyclonal antibodies to: FABP4 (Cloud-Clon Corp., Houston, TX, USA, at 1:1000 for 30 min), Omentin (Bioss Antibodies, Woburn, MA, USA, at 1:500 for 30 min) or CD36 (Invitrogen, Waltham, MA, USA, at 1:1000 for 30 min) or mouse monoclonal antibody to CD68-clone PGM1 (Agilent, ready to use for 20 min); (2) EnVision^®+ Dual Link System-HRP (Agilent Technologies, Inc., Santa Clara, CA, USA, dextran polymer conjugated with horseradish peroxidase and affinity-isolated goat anti-mouse and goat anti-rabbit immunoglobulins) (Agilent, K4065) for 20 min; (3) DAB+ substrate-chromogen solution (1 mL of substrate buffer solution containing hydrogen peroxide and 20 µL of 3,3′-diaminobenzidine tetrahydrochloride chromogen solution) (Agilent) for 10 min; and (4) EnVision FLEX hematoxylin (Agilent) for 15 min.

For RNA-ISH, slides incubated at 60 °C were de-paraffinized in xylene. Slides were then kept in 100% ethanol twice for 3 min each and then air-dried following treatment with H₂O₂ for 10 minutes. Further, slides were rinsed and boiled in 1X Target Retrieval for 15 min. After rinsing slides in distilled water, Protease Plus treatment was given and then incubated with DLX1 probe (Advanced Cell Diagnostics, probe ID: 569601) for 2 h at 40 °C. Next, slides were washed and treated with Amp 1 for 30 minutes, Amp 2 for 15 min, Amp 3 for 30 min, and Amp 4 for 15 min, all steps were carried out at 40 °C in the HybEZ oven with two washes in 1× Wash Buffer. Slides were then treated with Amp 5 for 30 min and Amp 6 for 15 min at room temperature in a humidity chamber. Red color was developed by adding a 1:60 solution of Fast Red B: Fast Red A to each slide and incubating for 10 min. Finally, slides were treated with EnVision FLEX Hematoxylin (Agilent DAKO, K800821-2) and mounted using the same protocol as used for IHC slides.

To assess the ability of resveratrol to induce the apoptotic process, an ICC reaction was performed. The cells of the tested lines were plated on Millicell EZ SLIDES eight-well glass slides (Merck Millipore, Gernsheim, Germany) in the following amounts: EPP85-181P—1 × 10⁴ cells/well, EPP85-181RNOV—1 × 10⁴ cells/well, AsPC-1—1.5 × 10⁴ cells/well and H6c7—1.5 × 10⁴ cells/well. 24 h later, cells were treated with resveratrol at concentrations of 0, 25, 50 and 100 µM for 48 h. After this time, cells were fixed with methanol-acetone (1:1) for 10 min at 4 °C. The ICC reaction was performed on an Autostainer Link48 (Dako, Glostrup, Denmark). The following primary antibodies were used: Bcl-2 (Dako, Glostrup, Denmark), Bax (Santa Cruz Biotechnology, Dallas, TX, USA) and activated Caspase-3 (Cell Signaling Technology, Boston, MA, USA). Slides were first incubated with primary antibodies against Bcl-2 (ready-to-use), Bax (1:25) and activated Caspase-3 (1:400) for 20 min at room temperature, followed by 20 min with EnVision FLEX/HRP (Dako, Glostrup, Denmark). In the next step, the slides were incubated for 10 min with 3,3’-diaminobenzidine (DAB, Dako. Glostrup, Denmark). The slides were counterstained with EnVision FLEX Hematoxylin (Dako, Glostrup, Denmark) and sealed with coverslips in a mounting medium. The ICC reaction was assessed using a BX-41 light microscope (Olympus, Tokyo, Japan).

Slides from TMAs (4 μm-thick) were used for immunohistochemistry (IHC) reactions, which were performed using DakoAutostainer Link48 (Dako, Glostrup, Denmark). In order to deparaffinize, rehydrate and unmask the antigens the sections were boiled in EnVision FLEX Target Retrieval Solution (pH 9, 20 min, 97 °C; Dako) using the PTLink platform (Dako, Glostrup, Denmark). Afterwards, slides were incubated for 5 min with Envision Flex Peroxidase-Blocking Reagent (Dako, Glostrup, Denmark) to block endogenous peroxidase. As primary antibodies (20 min, RT), rabbit polyclonal antibodies against AMH (1:100, ab84952, Abcam, Cambridge, UK) were used. Next, slides were incubated with EnVision FLEX/HRP (20 min, RT), and the reaction was visualized (10 min, RT) with freshly prepared 3,3′-diaminobenzidine (DAB). Additionally, slides were counterstained for 5 min with EnVision FLEX Hematoxylin (Dako, Glostrup, Denmark). Finally, slides were dehydrated in ethanol (70%, 96%, absolute) and xylene, then mounted with Dako Mounting Medium (Dako, Glostrup, Denmark). Slides were evaluated using the Olympus BX41 light microscope (Olympus, Japan). Control tissues included the human prostate.

After 24 h, the lungs and trachea collected from the infected and mock-infected voles were fixed in 10% neutral phosphate-buffered formalin, paraffin-embedded, and stained with hematoxylin-and-eosin method for histopathological examination. The immunohistochemistry (IHC) was carried out by an automated immunostainer (Autostainer™ Link 48 Dako, Italy). Briefly, the antigen retrieval was performed with Proteinase K (Dako, Italy) for 3 min at room temperature (RT). Endogenous peroxidases were neutralized by incubating the sections with the EnVision FLEX Peroxidase-Blocking Reagent (Dako, Italy) for 10 min at RT. Sections were incubated with a primary monoclonal antibody against IA virus nucleoprotein (Clone 1331, BIODESIGN International, USA), applied at 1:500 dilution for 10 min at RT. The EnVision FLEX/HRP (Dako, Carpinteria, CA, USA) and the EnVision FLEX Substrate Buffer EnVision FLEX DAB+ were used as the detection system and chromogen, respectively. Sections were then counterstained with the EnVision FLEX Hematoxylin (Dako, Italy). The specificity of the immunostaining was verified by incubating some sections with PBS instead of the specific primary antibody.