Ab189513

Brains from wild-type and NSG1 KO mice were homogenized in 5mM CHAPS, 50mM Tris-HCl, 150mM NaCl, 1 × proteinase inhibitor with a Kontes microtube pellet pestle while kept on ice. Detergent-insoluble material was removed by centrifugation at 13,000 × g for 10 min. Aliquots of supernatants were mixed with SDS sample buffer (125 mM Tris-HCl, pH 6.8, 20% glycerol, 4% SDS, 0.02 percent bromophenol blue, and 125 mM dithiothreitol) and incubated at 50°C for 20 minutes. The samples were separated on a 10% NuPage Bis-Tris gel (Invitrogen) using a MES buffer system (Invitrogen), transferred to nitrocellulose membranes using the Trans-Blot Turbo Transfer System (Bio-Rad), blocked in Odyssey Blocking Buffer (LI-COR), and probed simultaneously with the primary antibodies: mouse anti-NSG1 (Santa Cruz; sc-390654, 1:1000) and rabbit anti-NSG2 (Abcam; ab189513, 1:1000) followed by incubation with the IRDye conjugated secondary antibodies: goat anti-mouse800CW (LI-COR; 926–32210, 1:10,000) and goat anti-rabbit680RD (LI-COR; 926–68071, 1:10,000). The Odyssey CLx infrared imaging equipment was used to detect infrared signals

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.