Anti aldh1l1

For immunofluorescent stains, we rinsed the cells and 3D cultures twice with PBS (phosphate buffered saline). Cells were then fixed through a RT 15–30 min incubation in fresh 4% paraformaldehyde aqueous solution (157–4, ElectronMicroscopy Sciences) followed by rinsing twice with PBS. Cells were permeabilized through incubation in 0.1% Triton X-100 in PBST (phosphate buffered saline with 0.1% tween®20) for 15 minutes at RT. Cell on-specific binding was blocked through overnight incubation in 3% human serum albumin in PBST at 4 °C. After a 24-hours incubation with the primary antibody solutions at 4°C, the cells were washed five times. The following antibodies (and dilutions) were used: anti-p-tau antibody (1:40, AT8, Thermo Scientific, MN1020); anti-PHF (1:1000, A gift from P. Davies, Albert Einstein College of Medicine); anti-GFAP antibody (1:500, Neuromap, N206A/8), anti-MAP2 antibody (1:200, Cell Signaling Technology, 4542); anti-CD68 (1:100, Cell Signaling, 76437); anti-cd11b (1:100, Life Technologies, NB110–89474); anti-S100 (1:400, Abcam, ab868); anti-S100A6 (1:200, Cell Signaling, D3H3W); anti-S100B (1:500, Abcam, Ab218515); anti-ALDH1L1 (1:200, EMD Millipore, MABN495).

Protein lysates (15–75 μg) were resolved on 12% Bis-Tris or 4–12% gradient Bis/Tris gels (Life Technologies) and the proteins were transferred to nitrocellulose membranes (Bio-Rad). For amyloid-ß western blot analysis, the membranes were cross-linked with 0.5% glutaraldehyde solution before blocking. Western blot results were visualized using enhanced chemiluminescence (ECL). Signals were captured using ChemiDoc imaging system (Bio-Rad) and quantified using ImageJ software (NIH). The following primary antibodies were used (dilutions): anti- Aβ (1:100, 6e10; Signet); anti-Tuj1 (1:200, Abcam, ab24629); anti-MAP2 (1:200, Cell Signaling, 4542); anti-GFAP (1:100, Neuromap, N206A/8); anti-p-tau AT8 (1:30, Thermo Scientific, MN1020), p-tau PHF-1 (1:200, Abcam, ab66275), CD68 (1:200, BD Bioscience, 556059), CD11b (1:200, EMD Millipore, MM_NF-MABF515), anti-ALDH1L1 (1:200, EMD Millipore), Cy5 anti-mouse secondary antibody (1:400, Jackson Immunoresearch, 715-175-150).