Quantitative Analysis of Brain Engraftment

We acquired epifluorescence images using a BZ-X800 Microscope (Keyence). Whole brain tiled images were taken at 10× magnification and stitched using Keyence Analyzer software prior to image export. Representative images were taken at 10×. We analyzed images in Fiji (https://imagej.net/Fiji), adjusting for brightness and black values, but we performed no other image math. Engraftment renderings were made in Fiji (example, Fig. S1 A). The 488/GFP channel was processed by subtracting background, adjusting threshold level, analyzing particles (size = 0–0.1 in²; circularity = 0.5–1), making a mask of GFP+ cells similar to masking strategy used by Kozlowski et al. (Kozlowski and Weimer, 2012 (link)), converting into ultimate points, and overlaying onto the DAPI channel. The entire brain was then outlined using ImageJ’s “polygon sections” tool and anything falling outside of this brain outline was excluded (example, Fig. S1 J). Representative confocal images were captured using a Leica SP8 confocal microscope equipped with a Diode 405 laser, HyD sensors, 40×/1.30 oil lens, and controlled with Leica LAS X v.3.5.2 software. Z-stack processed using maximum intensity projection function in ImageJ.