Fv500 confocal microscope

Cells grown in 8-well chambers were fixed in 4% formalin and blocked. They were then probed with anti-ki67 antibodies (Abcam) or anti-human p21 antibodies (Abcam). Alexa Fluoro antibodies (Invitrogen) were subsequently used. The nuclei were stained using 4’,6-diamidino-2-phenylindole (DAPI, Invitrogen). Ki67- or p21- positive cells were counted using ImageJ (16 (link)). To measure superoxide levels, dihydroethidium (DHE, Invitrogen) was used. After fixation, DHE (10 μM) was added to CYR61- or control-transfected fibroblasts for 15 min, and then stained with DAPI. Fluorescence was detected using an Olympus FV-500 confocal microscope and photographs were taken at 400x.

For frozen sections, fixed embryos were rinsed in PBS and incubated in 30% sucrose solution prepared in PBS until embryos sunk to the bottom of the tube. This was followed by incubation in 50/50 mix of 30% sucrose solution and optimum cutting temperature (OCT) compound (Tissue-Tek) at 4°C overnight. Embryos were then embedded in OCT and frozen using an isopentane/dry ice bath. The following primary antibodies were diluted in blocking buffer (10% donkey serum, PBS, 0.05% Tween-20) and used to stain frozen sections overnight at 4°C: anti-VEGFR2 (1:200, R&D, #AF644), anti-ERG (1:100, Abcam, #ab110639), anti-GFP (1:500, Aves labs, #GFP-1020), and anti-Cre (1:500, Millipore, #MAB3120). Slides were washed in PBS containing 0.05% Tween 20 (PBS-Tween) with three changes of washing buffer, 10 min each, and then incubated with Alexa-conjugated 2° antibodies, diluted 1:300 in blocking buffer for 2 hours at room temperature. After washing in PBS-Tween, sections were mounted using a 1:1 mixture of methanol and glycerol (v/v) to extinguish the native GFP and tdTomato fluorescence. Slides were imaged using Olympus FV500 confocal microscope. Each experiment was repeated at least three times, using 3 independent embryos.