For the quantification of various tau species in brain homogenates, different AlphaLisa assays were developed according to manufacturer instructions (Perkin Elmer). Acceptor beads were conjugated to either the HT7 anti-human tau antibody (Thermo Fisher Scientific, MN1000) or the TAU-13 anti-human tau (Abcam, B11E8). Detection antibodies pSer202/pThr205 (AT8, Thermo Fisher Scientific, MN1020), pThr181 (AT270, Thermo Fisher Scientific, MN1050), pThr231 (AT180, Thermo Fisher Scientific, MN1040), human tau (HT7, Thermo Fisher Scientific, MN1000), pThr212 (Santa Cruz Biotechnology), MC1 (kindly provided by Prof. Peter Davies), pSer409 and pSer396 (AC Immune) were either purchased with biotin tag or conjugated to biotin using EZ-Link™ Sulfo-NHS-Biotin (Thermo Fischer Scientific). Brain total homogenates were pre-diluted in PBS to obtain a 5 µg/µl stock concentration. Dilutions of all reagents were made with Alpha Assay buffer (PerkinElmer). Reagents were added in a 384-well white OptiPlate (PerkinElmer) to a final volume of 50 µl. 5 µl of total brain homogenate (final concentration in an assay: 0.5 µg/µl), 10 µl of biotinylated detection antibody (final concentration: 10 nM), 10 µL of tau acceptor beads conjugate (final bead concentration: 2.5 µg/ml) were incubated 1 h at RT, before the addition of 25 µl of donor beads (final bead concentration: 25 µg/ml) and a further incubation at RT for 30 min (protected from light). The plate reading was performed using EnSpire Alpha instrument and analysis using EnSpire Workstation version 3.00.