Donkey antigoat and donkey antirabbit

Wild-type and NSG1 KO mice were anesthetized and perfused with phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA). Brains were removed and immersed in 4%PFA, 20% sucrose, and 30% sucrose, each for 12–24 hours. Sagittal sections were sliced on a sliding knife microtome (American Optical). Free-floating sections were permeablized and blocked simultaneously in 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MO) and 10% donkey serum (Millipore-Sigma) for 1 hour in PBS. Sections were stained with goat anti-NSG1 (Thermofisher; PA5-37939, 1:1000) rabbit anti-NSG2 (Abcam; ab189513, 1:500) in 0.25% Triton and 5% donkey serum in PBS overnight at 4°C. Sections were washed with three times in PBS followed by secondary antibody labeling (donkey anti-goat and donkey anti-rabbit (1:1000); Thermofisher) in the same buffer as primary antibodies. Sections were then mounted to microscopy slides (Superfrost plus, Fisher Scientific) immersed in Fluoromount-G, and imaged on a slide-scanning microscope (Zeiss Axion Scan.Z1) with Colibri 7 LED light source following standard fluorescence calibration on control brain sections to ensure proper dynamic range of signals. Imaging conditions were then maintained on NSG1 KO brains.