Dako mayer s hematoxylin lillie s modification histological staining reagent

Immunohistochemistry was performed using the Dako EnVision^™ + Dual Link System-HRP (DAKO). For antigen-retrieval, sections were heated in citric acid buffer (pH 6.0) at 100°C for 10 min using Lab Vision PT Module (Thermo Fisher Scientific Inc.). The sections were treated with 0.3% hydrogen peroxide in methanol for 30 min at room temperature to block endogenous peroxidase activity and incubated with 5% normal goat serum in T-PBS for 1 hour at room temperature to block non-specific protein binding sites. They were then incubated at 4°C with the primary antibodies diluted at 1:40 (anti-human CD31), 1:50 (anti-Ajuba and CD45), 1:200 (anti-YAP), 1:400 (anti-Survivin), 1:2 (anti-GLUT1) and 1:16 (anti-CD68) in T-PBS. After overnight incubation, the sections were incubated with EnVision reagents for 1 hour at room temperature and treated with Dako Liquid DAB+ Substrate Chromogen System (Cat No. K3468, DAKO) to visualize the reaction products. Sections were counterstained with Dako Mayer’s Hematoxylin (Lillie’s Modification) Histological Staining Reagent (DAKO). Digital immunohistochemical images were captured on an Olympus IX71 inverted microscope equipped with a MicroFire^™ camera and PictureFrame^™ 1.0 software for Macintosh (OPTRONICS) and Photoshop CS2 software (Adobe) on a Windows 7 computer.

Tissue sections were de-paraffined using xylene and a graded series of ethanol. For antigen-retrieval, sections were heated in citric acid buffer (pH 6.0) at 100˚C for 15 minutes. Non-specific background staining of endogenous peroxidase was treated with 0.3% hydrogen peroxide for 30 minutes, and sections were blocked with 3% bovine serum albumin in PBS (pH 7.4) for 1 hour at room temperature. Sections were then incubated at 4˚C with the primary antibody (Major Resources Table). After overnight incubation, the sections were incubated with HRP conjugated secondary antibody for 1 hour at room temperature and treated with Dako Liquid DAB+ Substrate Chromogen System (GV825, Agilent Dako) to detect the reaction products. Finally, the sections were counterstained with Dako Mayer’s Hematoxylin (Lillie’s Modification) Histological Staining Reagent (S3309, Agilent Dako). For negative controls for the antibodies, IgG isotype controls, negative tissue controls and endogenous tissue background controls were used.

Tissue sections were de-paraffined using xylene and a graded series of ethanol. For antigen-retrieval, sections were heated in citric acid buffer (pH 6.0) at 100°C for 15 minutes. Non-specific background staining of endogenous peroxidase was treated with 0.3% hydrogen peroxide for 30 minutes, and sections were blocked with 3% bovine serum albumin in PBS (pH 7.4) for 1 hour at room temperature. Sections were then incubated at 4°C with the primary antibody (Major Resources Table). After overnight incubation, the sections were incubated with HRP conjugated secondary antibody for 1 hour at room temperature and treated with Dako Liquid DAB+ Substrate Chromogen System (GV825, Agilent Dako) to detect the reaction products. Finally, the sections were counterstained with Dako Mayer’s Hematoxylin (Lillie’s Modification) Histological Staining Reagent (S3309, Agilent Dako). For negative controls for the antibodies, IgG isotype controls, negative tissue controls and endogenous tissue background controls were used.