Leo 1550 scanning electron microscope

Three littermate gender matched pairs of 6-week-old mice with each pair consisting of one mutant and one control animal (total of 6 animals; 4 males and 2 females) were sacrificed using carbon dioxide euthanasia, and then perfused with 20 ml of PBS with 200 USP units of heparin using a solution that was pre-warmed to 37°C. The perfusion mixture was injected through the left ventricle and an incision made in the right atria to release the injected fluid. A successful perfusion was indicated by a slow clearance of red blood cells from the kidney, as indicated by a change in the color of the kidney tissue. After flushing out the blood, the mouse was injected with 10 ml of corrosion casting solution (Batson’s No. 17 Corrosion Kit) at 1 ml/minute through the left ventricle of the heart. The casting solution was left to cure for at least 2 hours at room temperature, then the organ was digested with 20% KOH and washed with water until the tissue was fully dissolved, leaving only the corrosion cast. Select regions of a cast were then mounted and sputter coated with 2nm of iridium using a Leica ACE600. Samples were then imaged using a Zeiss LEO 1550 Scanning Electron Microscope.