Horseradish peroxidase conjugated anti mouse antibody

Bacterial cells cultured in the desired medium were collected by centrifuge and opened by sonication after resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA). Bacterial lysates were normalized to the same amount of total proteins before being treated with the SDS-PAGE loading buffer. Proteins were separated in 12% SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad) in a semi-dry electroblot cell (Bio-Rad). The HA-tagged and FLAG-tagged proteins were reacted with mouse monoclonal anti-HA and anti-FLAG antibodies, respectively (Sigma), detected with a horseradish peroxidase-conjugated anti-mouse antibody (Bio-Rad), and then visualized using ECL western blotting substrate (Pierce). A magnesium transporter protein CorA was used as the control and detected with a mouse multi-clonal antibody made in our laboratories. Relative protein amount was measured by ImageJ¹.

Brains and spinal cords were homogenised in 2 ml/g buffer [25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 5 mM sodium pyrophosphate, 10 mM β-glycerophosphate, 30 mM sodium fluoride, 10 mM sodium vanadate, 10 mM PMSF and one tablet of complete protease inhibitor cocktail (Roche) per 20 ml buffer], followed by a 30 min centrifugation at 150,000 g. The supernatants from at least three mice per group were diluted 1:150 (30–60 ng protein/ml) in reagent diluent (R&D Systems) and incubated overnight at 4 °C in 96-well ELISA plates, which were blocked with phosphate-buffered saline (PBS) containing 0.2% Tween-20 and 3% bovine serum albumin (BSA) for 1 h at 37 °C. To assess human Tau transgene expression levels, the supernatants were then incubated with human-specific [27 ] anti-Tau antibody HT7 (1:6,000, Fisher Scientific) for 2 h, followed by horseradish peroxidase-conjugated anti-mouse antibody (1:50,000, Bio-Rad) for 1 h. Following a colorimetric reaction, the plates were read on a Tecan plate reader. Purified recombinant Tau and brain lysates of heterozygous mice transgenic for human mutant P301S Tau were used as standards.