Mouse anti phospho tau at8

Western blots were performed on tissue homogenates as described previously^{59 (link)}. Clarified supernatants were, diluted to matching protein concentration (23 μg total) and heated with Laemmli loading buffer (BioRad, Hercules, CA; 95 °C, 5 min) and separated on a 12% TGX gel (Bio-Rad, #161-0180) by SDS-PAGE. Prior to protein transfer to the blotting membrane all protein bands were visualized using a ChemiDoc^TM MP system (BioRad) and total band densities were obtained via Image Lab v5.2. Proteins were then transferred onto a PVDF membrane, blocked in 5% w/v skim milk powder (in 0.1% v/v TBS-T; 1 h, at 23 °C), then incubated with a mouse anti-Phospho-Tau (AT8; Thermo Scientific, Waltham, MA, #MN1020) overnight at 4 °C. Membranes were blocked at 4 °C and incubated with a rabbit anti-NRF2 antibody (Thermo Fisher Scientific, #710574) for 3 h, at 23 °C and then incubated with either an anti-mouse or anti-rabbit HRP-conjugated secondary antibody (dilution 1:5000 v/v; 1 h at 23 °C). Protein bands were visualized using Luminata Forte HRP substrate (Millipore, Billercia, USA) and imaged on a ChemiDoc^TM MP system (BioRad).

Medulla, mesencephalon, and entorhinal cortex paraffin-embedded sections were dewaxed in xylene and rehydrated. Antigens were retrieved using 80% formic acid for 20 min at room temperature. Before primary antibody incubation, samples were incubated for 20 min with 1% BSA diluted in 0.01 M phosphate saline buffer (PBS) containing 0.1% Triton X-100 (PBS-T). Primary antibodies (rabbit anti-HDAC6, 1:50, GeneTex or rabbit anti-phospho-HDAC6, 1:50, GeneTex and mouse anti-α-synuclein LB509, 1:500, Abcam or mouse anti-phospho-tau AT8, 1:150, Thermo Fisher Scientific or mouse anti-β-amyloid, 1:2000, Wako) in 1% BSA diluted in PBS-T were incubated overnight at room temperature. After washing, samples were incubated for 2 h at room temperature with highly pre-adsorbed secondary antibodies, in particular Alexa Fluor^® 568 goat anti-mouse (Molecular Probes) and Alexa Fluor^® 488 donkey anti-rabbit (Molecular Probes). TO-PRO^®-3 (1:1000 for 10 min; Molecular Probes) was used for nuclei counterstaining. Finally, samples were mounted using 0.01 M PBS-glycerol (1:2) and examined with a TCS SP8 confocal microscope Leica equipped with an Argon laser coupled with a hybrid detector, a diode-pumped solid-state laser coupled with a photomultiplier tube and a helium/neon mixed gas laser coupled with a hybrid detector.