Envision flex hrp

Formalin fixed, paraffin embedded lung tissues were cut into 5 μm thick sections, stained with hematoxylin/eosin and evaluated by light microscopy. Immunohistochemical staining was performed to detect EGFP expressed in tumor cells. Slides were deparaffinized and rehydrated in phosphate buffered saline solution (10 mM, pH 7.2). The tissue epitopes were demasked using the automated water bath heating process in Dako PT Link (Dako, Glostrup, Denmark); the slides were incubated in pH 6.0 citrate retrieval buffer at 98°C for 20 minutes. The slides were subsequently incubated for 60 minutes at room temperature with the primary rabbit polyclonal antibody against GFP (Abcam, anti-GFP, ab290) diluted 1:500 in Dako REAL antibody diluent (Dako, Glostrup, Denmark) and immunostained using anti-mouse/anti-rabbit immuno-peroxidase polymer (EnVision FLEX/HRP, Dako, Glostrup, Denmark) for 30 minutes at room temperature, according to the manufacturer’s instructions. For visualization, the slides were reacted with diaminobenzidine substrate-chromogen solution (DAB, Dako, Glostrup, Denmark) for 5 minutes. Finally, the slides were counterstained with hematoxylin.

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 were deparaffinized and rehydrated in phosphate buffered saline solution (10 mM, pH 7.2). The tissue epitopes were demasked using the automated water bath heating process in Dako PT Link (Dako, Glostrup, Denmark); the slides were incubated in TRIS-EDTA retrieval solution (10mM TRIS, 1mM EDTA pH 9.0) at 98°C for 20 minutes. The slides were subsequently incubated for 1 hour at room temperature with the primary mouse monoclonal antibody against PD-1 (Abcam, [NAT105]: AB52587) and rabbit monoclonal antibody against PD-L1 (Abcam [EPR1161(2)]: AB174838) diluted 1:100 in Dako REAL antibody diluent (Dako, Glostrup, Denmark) and immunostained using anti-mouse/anti-rabbit immuno-peroxidase polymer (EnVision FLEX/HRP, Dako, Glostrup, Denmark) for 30 minutes at room temperature, according to the manufacturer's instructions. Color reaction was developed with diaminobenzidine substrate-chromogen solution (DAB, Dako, Glostrup, Denmark) for 5 minutes. Finally, the slides were counterstained with haematoxylin, mounted and reacted for 5 minutes with diaminobenzidine substrate-chromogen solution (DAB, Dako, Glostrup, Denmark) for visualization. PD-1 and PD-L1 positivity of lymphocytes in the tonsil was used as a positive control, same tissue with omitting of the primary antibody served as negative control.

One aortic valve from an AS patient was processed with this methodology. After surgical sample collection, valve leaflets were fixed in 10% buffered formalin for 24 h and processed for paraffin embedding using standard procedures for histological and IHC analysis. The specimens were sectioned at 4 µm thickness and stained with haematoxylin–eosin (H&E). Decalcification with a 10% EDTA solution was done if needed.
For IHC analysis, 3 µm-thick sections were obtained, and immunostaining was performed using the rabbit monoclonal antibody anti-CD3 (1:150) (Abcam ab16669, Cambridge, UK) with the Envision FLEX/HRP system (Dako, Glostrup, Denmark). For IHC staining, the secondary antibody (Envision FLEX/HRP) was used for 30 min at room temperature, followed by 3,3′-diaminobenzidine (DAB) staining (Dako, Glostrup, Denmark) before being counterstained with Harris haematoxylin. Human lymph node was used as a positive control for CD3 antibody and staining in the absence of the primary antibody was used as a negative control. IHC images were acquired using a Leica microscope (Leica Microsystems, Wetzlar, Germany) and a Thunder Imager microscope (Leica Microsystems, Wetzlar, Germany), respectively.

Immunohistochemical (IHC) staining was performed incubating 4 µm FFPE sections in 10 mmol/L citrate buffer (pH 6.0) at 120°C for 5 minutes for antigen retrieval. Endogenous peroxidase was neutralized using EnVision FLEX Peroxidase‐Blocking Reagent (Dako) for 10 minutes. Tissue sections were blocked with 3% bovine serum albumin and incubated with the primary antibodies overnight at 4°C. Primary antibodies used were FRMD6 (Sigma‐Aldrich), ZEB1 (Sigma‐Aldrich), HTR2B (Sigma‐Aldrich), AE1AE3 (Thermo Scientific), CDX2 (Novus Biologicals), SRSF3 (Abcam) and SERPINA1 (Sigma‐Aldrich). After incubation with the EnVision FLEX + mouse or rabbit linker (Dako), EnVision FLEX/HRP (Dako) was used as the secondary antibody for 1 hour at room temperature, followed by 3,3'‐diaminobenzidine (DAB) staining (Dako). CMS molecular classification was performed according to Thrin et al,14 analysing the intensity and content of FRMD6, ZEB1, HTR2B, AE1AE3 and CDX2. SRSF3 and SERPINA1 tumoral epithelial expression was categorized as high (intense staining) and low expression (moderate and negative staining). Individual cores were scored by trained pathologists (CVP and SGL).