Axiocam hrc system

After retroviral transduction 5 x 10³ transduced Rat-1 cells were suspended in ‘top-agar’, DMEM supplemented with 10% FCS and 0.25% bacto-agar (DIFCO Laboratories, Detroit, USA), and stacked in six-well plates filled with DMEM supplemented with 10% FCS and 0.5% bacto-agar (2 ml per well). After 15 days incubation at 37°C and 5% CO₂ colonies were counted. The focus-formation assays were performed in 24-well plates. 4 x 10⁴ transduced Rat-1 cells/well were plated. Unstained foci were photographed at day 15 using an AxioCam HRc system (Zeiss, Goettingen, Germany) with 10x magnification.

Samples were embedded in OCT compound and snap-frozen. Sections (5-μm thick) were fixed in 4% paraformaldehyde for 15 min. For immunofluorescence staining, frozen sections were blocked with 1% bovine serum albumin and 5% fetal bovine serum. Sections were immunostained for Tie2 using goat polyclonal anti-Tie2 (Santa Cruz Biotechnology, CA, USA) antibodies and for the EC and hematopoietic cell markers CD31 and CD45 using fluorescein isothiocyanate (FITC)-conjugated anti-CD31 and allophycocyanin (APC)-conjugated anti-CD45 antibodies, respectively (BD Pharmingen, CA, USA). Immunohistochemistry images were taken using an Axioskop 2 Plus direct microscope (Zeiss, Oberkochen, Germany) equipped with a Radiance 2100 three-laser confocal device (Bio-Rad, Segrate, Italy) and a W-PI × 10/0.23 or Plan-Neofluor × 20/0.5 numerical aperture objective lens (Zeiss). Images were captured using the AxioCam HRc system and Axiovision 3.1 version 4.4 software (Zeiss). Fluorescent signals from the individual fluorophores were sequentially acquired from single optical sections and were analyzed using Paint Shop Pro X (Corel, Ottawa, Canada).