Tetramethylbenzidine solution

For ELISA of misfolded SOD1, motor neurons on day 7 were harvested and dissolved in buffer containing 1% Triton-X, 0.5% deoxycholate, 50 mM Tris–HCl, 1 mM EDTA, 0.1% SDS, 150 mM NaCl, 0.1% sodium deoxycholate, protease inhibitor (Roche), and phosphatase inhibitor (Roche). Samples were centrifuged at 13,000×g for 15 min at 4 °C. Then, 96-well plates (Thermo Fisher Scientific) were coated with 3 μg/ml MS785 antibody in 0.05 M sodium carbonate buffer at 4 °C overnight. After washing and blocking with TBS-T containing 1% BSA, 200 μg protein/100 µl of samples were added, and incubation was carried out for 2 h at room temperature. Recombinant mutant SOD1 protein (G93A) was used to obtain a standard curve. For detection, the plates were incubated with 3 μg/ml anti-SOD1 antibody (ENZO), followed by sheep anti-rabbit IgG F(ab)’2 fragment linked to horseradish peroxidase (1:3000; GE Healthcare). After incubation with tetramethylbenzidine solution (BD Bioscience) at room temperature for 30 min, absorbance at 450 nm was measured by VersaMax (Molecular Device, Sunnyvale, CA, USA).

96-well plates (Greiner, Frickenhausen, Germany) were coated with 3 μg/ml TOC1 antibody in 0.05 M sodium carbonate buffer at 4 °C overnight. After washing and blocking with TBS-T containing 1% BSA, 100 μl of cultured medium was added, and incubation was carried out for 2 h at room temperature. Recombinant oligomeric tau protein was used to obtain a standard curve. For detection, the plates were incubated with 2 μg/ml affinity-purified rabbit polyclonal anti-human tau antibody, followed by sheep anti-rabbit IgG F(ab)’2 fragment linked to horseradish peroxidase (1:3,000; GE Healthcare). After incubation with tetramethylbenzidine solution (BD Bioscience) at room temperature for 30 min, absorbance at 450 nm was measured by VersaMax (Molecular Devices, Sunnyvale, CA). The anti-human tau antibody was raised against tau amino-terminus polypeptide (amino acids 19–33, according to the longest human tau isoform) by immunization of two rabbits.

Hippocampal human phosphorylated tau protein was evaluated by ELISA assay using anti-phosphorylated antibody (AT8, #. MN1020, Thermo Fisher Scientific). Microtiter plates were coated with 3 µg/ml AT8 antibody in 0.05 M sodium carbonate buffer (pH 9.4) at 4 °C overnight. After washing and blocking with TBS-T containing 1% BSA, 100 µl of diluted mouse hippocampus RAB-HS soluble fractions (the protein concentration of each sample was adjusted) was added, and the incubation was carried out for 2 h at room temperature. Serial dilutions of collected FTLD-tau mouse brain cortical lysates ranging from 1:100 to 1:102,400 were used as positive control. For detection, the plates were incubated with 2 μg/ml rabbit anti-human tau protein antibody (#. A0024, DAKO), followed by sheep anti-rabbit IgG F(ab)′2 fragment linked to horseradish peroxidase (GE Healthcare) at 1:3000 dilution. After incubation with tetramethylbenzidine solution (BD Bioscience) at room temperature for 30 min, absorbance at 450 nm was read on an automated plate reader (Model 353; Thermo Fisher Scientific). The amount of phosphorylated tau protein of positive control was defined as 1000 units, and unknown titers of samples were determined by interpolation from a standard curve generated by positive control standards of known dilution. All samples were analyzed in duplicate.