Batson s no 17 plastic replica and corrosion kit

One fetus from each ewe was corrosion cast using 7 ml of resin from Batson's No. 17 Plastic Replica and Corrosion Kit (Polysciences, Inc., Warrington, PA, USA), injected via the umbilical artery in a retrograde direction in accordance with the manufacturer's protocol. After perfusion, the resin-cast whole fetus was allowed to set on ice for 2–3 h, followed by immersion in maceration solution (20% potassium hydroxide) for 12–18 h at 50°C to remove the surrounding soft tissue. Thereafter, the vascular cast was rinsed, dried, and stored at room temperature. The surface area and volume of the resin casts of each fetal kidney were quantified using a Nanotom high-resolution computed tomography scanner (GE Sensing and Inspection Technologies, Wunstorf, Germany). X-ray slices were taken every 40 μm throughout the cast, averaging 2000 slices/fetus. Using Nanotom software, these images were stacked and reconstructed in 3D (see Supplemental Movie S1) to provide quantitative volumetric data and to calculate the surface area of each cast.

Heart resin casts were produced from E17.5–18.5 embryos using the Batson’s No. 17 Plastic Replica and Corrosion Kit (Polysciences, Inc) prepared according to manufacturer instructions. Embryos were dissected from the uterus and decapitated to allow flow of the resin through the heart. The pericardial cavity was exposed and a 1 cc insulin syringe was used to inject resin into the right, then left ventricle for each embryo. The embryo body was left on ice for 3 h to overnight in a refrigerator to allow the resin to harden. Tissue was then removed from the cast through immersion in Maceration Fluid from several hours to overnight, followed by washing in distilled water. Casts were dissected to reveal the pulmonary arteries and veins on the dorsal side of the heart, then photographed using an Olympus SZ61 microscope and SeBaCam5C camera. Casts were injected with blue-dyed resin in the right ventricle and red-dyed resin in the left ventricle, but the dyes mixed unpredictably due to the presence of the foramen ovale connecting the atria. Because of this, meaningless colour variation in the vessels can be seen even in the greyscale images shown.

Mice were perfused with maximal vessel dilation as described above. Brain arterial vasculature was then casted using a Batson’s No 17 Plastic Replica and Corrosion Kit (Polysciences, Inc, Warrington, PA, USA). Briefly, 1 ml of Batson’s 17 was infused through the thoracic aorta. After fully curing, the brain tissue was removed using maceration solution, and the cerebral vasculature including the pial collateral regions were carefully persevered for emission scanning electron microscopy. The vasculature was observed under a Zeiss Supra 25 Field emission scanning electron microscope. Images of pial collateral and DMAs were saved for analysis of primary cilia and endothelial cell morphology. To measure the orientation of collateral endothelial cells, we use Photoshop to draw a line coordinate with the collateral axis and a second line coordinate with the endothelial cell axis (see Figure 2, panel C), and the angle formed was measured.