Sig 39860

The following antibodies were used for the indicated dilutions. For immunoblotting: rabbit anti-NGLY1 (1:100; ATLAS ANTIBODIES; HPA036825), mouse anti-GAPDH (6C5; 1:5000; Millipore; MAB374), and HRP-conjugated antibody (1:10,000; CST; 7076S). For immunostaining: rabbit anti-GFAP (1:2000; Abcam; ab7260), goat anti-IbaI (1:1000; Abcam; ab5076), rabbit anti-GSTP1 (1:300; GenTex; GTX112695), rabbit anti-NeuN (1:3000; Abcam; ab177487), and mouse anti-NeuN (1:1000; BioLegend; SIG-39860).

After clearing, the brain was rinsed with PBST (1 × PBS with 1% Triton X-100, 0.02% sodium azide) at RT for 3-4 times. The brain was then embedded in 4% agarose (Sigma-Aldrich), cut including the neural probe into blocks of 1 mm (length) ×2 mm (width) ×1 mm (height). The brain tissue was incubated with mouse anti-FOX3 (1:500, SIG-39860, BioLegend), rat anti-GFAP (1:300, 13-0300, Invitrogen), and rabbit anti-Iba1 (1:300, PA5-27436, Invitrogen) for 4 days at RT. After incubation, brain tissue was rinsed in fresh PBST to 3 times before its overnight incubation at RT. The brain tissue was subsequently incubated with donkey anti-mouse Alexa Fluor 488 (1:300, Invitrogen), donkey anti-rat Alexa Fluor 594 (1:300, Invitrogen), goat anti-rabbit Alexa Fluor 647 (1:300, Invitrogen) for 3 days at RT. Then this brain tissue was rinsed with PBST up to 3 times at RT. Finally, this brain tissue was incubated in a refractive index matching solution for 30 min, and then images were scanned using a fluorescent microscope. The minimum animal numbers of each experiment (n = 6) were determined based on previous publications^{14 (link),53 (link)}.