Anti tubb3

Human SH-SY5Y-derived Flp-In host cells19 (link) were cultivated in DMEM-F12 containing 10% FBS, 500 μg/mL of geneticin (G418), and 5 μg/mL of blasticidin. The SH-SY5Y host cells were used to generate and maintain the ΔK280 tau_RD-DsRed line as described. SH-SY5Y tau_RD-DsRed cells were seeded with all trans-retinoic acid (10 μM; Sigma-Aldrich Co) in six-well (1×10⁵/well) plates.
On the next day, the cells were treated with Congo red (20 μM) or TCM extracts (NH008, NH014, NH016, NH021, NH027, NH034, and NH037; 500 μg/mL) for 8 hours. The tau_RD-DsRed expression was induced with Dox (1 μg/mL) for 1 week. The cells were then fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked in 3% bovine serum albumin, and stained with primary anti-TUBB3 (neuronal class III β-tubulin) antibody (1:1,000; Covance, Princeton, NJ, USA) at 4°C overnight and secondary anti-rabbit Alexa Fluor^® 555 antibody (1:500; Molecular Probes, Thermo Fisher Scientific) at room temperature for 3 hours. The total neurite outgrowth of untreated/treated cells was assessed using Metamorph Microscopy Automation and Image Analysis Software (Molecular Devices LLC).