Cy5 anti mouse secondary antibody

Primary hippocampal neurons were prepared in 24-well plates as described above. Growth medium was removed and replaced with prewarmed minimal essential medium (MEM). 2μg total DNA of mixture of INTRSECT and recombinase-expression plasmids (YFP-IRES-FlpO, BFP-IRES-Cre, mCherry-IRES-Cre) was mixed with 1.875 μL 2M CaCl2 (final Ca²⁺ concentration 250 mM) in 15 μL H2O. To DNA-CaCl2 we added 15 μL of 2 × HEPES-buffered saline (pH 7.05). After 20 min at room temperature (20–22°C), the mix was added dropwise into each cell culture well and transfection proceeded for 45–60 min at 37°C, after which each well was washed with 3 × 1 mL warm MEM before the original growth medium was returned. After 5 d expression, coverslips were fixed for 5 min at RT in 4% PFA. TVA-mCherry samples were rinsed twice in 1x PBS, then mounted on slides with Fluoromount-G (ThermoFisher). oG samples were fixed with 4% PFA, stained with anti-rabies glycoprotein primary antibody (abcam, 1:500) overnight at 4°C, and stained with Cy5 anti-mouse secondary antibody (Jackson ImmunoResearch, 1:500) for 3 hours prior to being mounted. Images were obtained on a Leica confocal microscope using 40 × and 63 × objectives.

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).