Transplanted mice were terminally anesthetized with an ip overdose of pentobarbital (150 mg/kg) and perfused transcardially with heparin saline (0.1% heparin in saline) followed by paraformaldehyde (4%) 4 months post grafting. Brains were removed, postfixed in 4% paraformaldehyde for 12 h, equilibrated in 20% sucrose/PBS solution, and then sectioned coronally at 40-µm using a cryostat. For immunofluorescence staining, tissue sections were incubated with blocking buffer (PBS, 10% NDS) containing 0.1% Triton for 10 min. Cells were then incubated overnight at 4 °C with primary antibodies diluted in PBS containing 2% NDS. After rinsing with PBS, samples were incubated with fluorescent dye-labeled secondary antibodies in PBS containing 2% NDS for 30 min at RT. After rinsing with PBS, Hoechst 33342 (4 µg/ml) was used for counterstaining, and tissue sections were mounted onto slides with Fluoromount-G. Confocal analysis was performed using an Olympus DSU Spinning Disc Confocal on an IX81 inverted microscope, installed with MetaMorph software. Stereo Investigator image capture equipment and software were used for cell counting and estimation of total cell number in the graft using the optical fractionator workflow from every 12th section.
