Map2b

NDC- and PS1- derived four-week-differentiated neuronal cultures, plated on 12 mm glass coverslips, were washed with PBS and incubated in opti-MEM for 30 minutes before transiently transfecting with 2 μg tau-PP-YFP + 1 μg mCherry, tau-WT-YFP + 1 μg mCherry, tau-LM-YFP + 1 μg mCherry or 1 μg mCherry using Lipofectamine 2000 transfection reagent (1:2; DNA:Lipofectamine, Thermo Fisher). Cells were incubated in DNA/Lipofectamine/opti-MEM for 2 hours and washed with 500 μl PBS. Fresh media was added and the cells were incubated until fixation at 2 or 4 days post-transfection. Prior to fixation, live imaging was performed on days 1, 2, and 4 using a Leica upright fluorescent microscope. Fixed cells were imaged using a confocal microscope. β -III-tubulin (1:500; Covance) was used to label all neurites and Map2b (1:500; Sigma) was used to distinguish dendrites. Axons were defined as β -III-tubulin+/Map2b-; dendrites β -III-tubulin+/Map2b+. Neurite lengths were measured using the NeuronJ plugin for ImageJ. Statistics were performed and figures were created using Prism.

NDC and PS-1-derived cultures were fixed at room temperature with 4% paraformaldehyde (Lamp2 samples were post-treated with cold 100% methanol for 5 minutes at −20°C) 4 days post-transduction or transfection. Fixed cultures were blocked for 45 minutes at room temperature in blocking solution (3% bovine serum albumin (BSA), 0.3% Triton X-100, 1x phosphate buffered saline (PBS)), stained for 2 hours at room temperature or overnight at 4°C, and washed twice with PBS (5 minutes/wash). The primary antibodies used were Map2b (1:500; Sigma), β -III-tubulin (1:500; Covance), synapsin (1:2000; Abcam), lamp2 (1:500; DSHB). All secondary antibodies were diluted 1:1000 in blocking solution and applied for 45 minutes at room temperature. Antifade solution (Prolong, Thermo) was applied prior to mounting the coverslips to glass slides, in an attempt to preserve the immunofluorescent signal. Samples were imaged with a Leica confocal microscope and data collection and analysis was performed using ImageJ.