Ultraview confocal imaging system

For mitotic cell purity analyses, the mitotic cells were plated onto cleaned-up coverslips and fixed in 4% paraformaldehyde for 10 minutes. Then the coverslips were washed with PBS and permeabilized for 10 min with 0.5% Triton X-100 in PBS. The cells were incubated with Anti-Histone H3 (phospho S10) antibody (ab5176) (diluted 1:100) and after blocking with 3% bovine serum albumin for 1 h. The cells were then incubated with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594 ab150077, for Anti-Histone H3 (phospho S10) and Alexa Fluor® 488 ab150140, for gamma H2A.X phospho S139) (diluted 1:200). The DNA is stained with DAPI (blue). The immunofluorescence was analyzed under a Perkin-Elmer Ultraview Confocal Imaging System.
For DNA damage analysis, cells were pretreated as above and were incubated with Anti-gamma H2A.X (phospho S139) antibody (ab2893) (diluted 1:100) after blocking with 3% bovine serum albumin for 1 h. The cells were then incubated with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) (diluted 1:200). The DNA is stained with DAPI (blue). The immunofluorescence was analyzed under a Perkin-Elmer Ultraview Confocal Imaging System.

HUVECs were transfected with 25 pmol siRNA (FlexiTube siRNA, Qiagen) per well of a six-well plate using 2.5 ml RNAiMax (Invitrogen) according to the manufacturer's instructions. After 24 h, HUVECs were labelled with either PKH26 (red) or PKH67 (green) (Sigma-Aldrich) according to the manufacturer's instructions, and a ratio of 1:1 cells were coated on cytodex3 microcarrier beads (Sigma-Aldrich) for 24 h. Beads were embedded in a fibrin gel (2.5 mg ml⁻¹ fibrinogen (Sigma-Aldrich) in EBM-2 (Lonza) supplemented with 2% FBS and 50 mg ml⁻¹ aprotinin (Sigma-Aldrich)) with fibrinogen solution clotted with 1 U thrombin (Sigma-Aldrich) for 30 min at 37 °C. WI38 cells (25,000 cells per well), in EBM-2 supplemented with 2% FBS and 50 ng ml⁻¹ VEGF, were then plated on top of the fibrin layer. Sprouts were imaged 3–4 days later on a Nikon eclipse Ti confocal microscope with the PerkinElmer UltraVIEW Confocal Imaging System and PerkinElmer Volocity software.