Swine anti rabbit fitc

Asynchronously-growing cells were treated for 15 min with Colcemid (KaryoMax, Invitrogen), resuspended in 75 mmol/L KCl and spun onto poly-l-lysine-coated slides (Cytospin, Shandon). Following fixation in absolute methanol, the cells were permeabilised in KCM (120 mmol/L KCl, 20 mmol/L NaCl, 10 mmol/L Tris.Cl, pH 8.0, 0.5 mol/L EDTA and 0.01% Triton X-100) and rabbit anti-chicken CENP-I antibody used at 1:1000 [50 (link)]. To estimate the mitotic index, cells were spun onto poly-l-lysine-coated slides in growth medium, fixed with 2% paraformaldehyde (ThermoFisher, Waltham, MA, USA) in PBS before permeabilisation in 0.1% Triton X-100 in PBS. The rabbit anti-histone H3S10ph antibody was used at 1:100 (Upstate). Secondary antibody, swine anti-rabbit FITC (Dako), was used at 1:100.

For immunofluorescence, HUVECs were seeded on gelatin-coated glass cover slips plated in 24-well plates and transfected with corresponding siRNA amount as described above. The cells were grown overnight in ECGM with 10% FCS. After incubation in 0.5% FCS for 24 h, the cells were washed with PBS and fixed with 4% formaldehyde for 10 min at room temperature. Then, cells were incubated in the blocking and permeabilization buffer containing 2.5% BSA and 0.3% Triton X-100. Subsequently, the cells were incubated with primary antibodies overnight at 4 °C. After washing with PBS, cells were incubated with secondary antibodies conjugated with FITC or TRITC for 1 h at room temperature. Finally, the slides were washed and mounted with mowiol (Calbiochem, Germany). The primary antibodies were rabbit-anti-FoxO1 (Cell Signaling), goat-anti-Ang-2 (Santa Cruz) and mouse-anti-O-GlcNAc (Abcam). The secondary antibodies were swine anti-rabbit FITC (DakoCytomation), swine anti-rabbit TRITC (DakoCytomation, Glostrup, Denmark), goat anti-mouse-FITC (Sigma-Aldrich) and Donkey anti-goat FITC (Acris, OriGene Europe, Herford, Germany). Photos were taken by confocal laser scanning microscopy (Leica Microsystems, Germany). Quantification of the protein expression in immunofluorescence was performed using Image J (NIH, USA).