Gfp 1020

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.

Probe templates were amplified by PCR from cDNA. cRNA probes were generated using Digoxigenin RNA Labeling Mix. Brains were cryosectioned after 4% paraformaldehyde perfusion, post-fixed in 4% paraformaldehyde, and prehybridized for 1 hour. Probes were hybridized overnight at 65°C, and washed for stringency, then detected with sheep anti-Digoxigenin and developed using Cy3 Tyramide Signal Amplification kit, followed by anti-Gfp(Aves, GFP-1020) and mouse anti-CNP1(Millipore, MAB326), detected with appropriate Alexa-dye labeled secondaries. Slides were imaged using an UltraView Vox spinning-disk confocal microscope.