Ix71 inverted microscope

To prevent non-specific protein binding, frozen tissue sections were incubated with 5% normal goat serum (Cat No. 005-000-121, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) in PBS (pH 7.4) containing 0.05% Triton X-100 (T-PBS) for 1 hour at room temperature. After blocking, sections were incubated with primary antibodies (CD31, CD45, CD11b, Mac3, and VEC diluted at 1:10 in T-PBS; CD31 (SL-4), YAP, and GLUT-1, diluted at 1:200; Survivin, diluted at 1:400; Ki67, diluted at 1:500; and phospho-YAP, diluted at 1:2000 over night at 4°C and further with secondary antibodies [Alexa Fluor 488 or 594-conjugated goat anti-rabbit IgG (H+L) or anti-rat IgG (H+L) (Life Technologies)], diluted at 1:100 in T-PBS for 2 hours at room temperature. Following three PBS washes slides were coverslipped with Dako Fluorescence Mounting Medium (Cat No. S3023, DAKO). Digital fluorescence images and phase-contrast microscopic photos were captured on an Olympus IX71 inverted microscope equipped with a MicroFire^™ camera and PictureFrame^™ 1.0 software for Macintosh (OPTRONICS, Goleta, CA, USA) and Photoshop CS2 software (Adobe, San Jose, CA, USA) on a Windows 7 computer.

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.