Anti phospho tau antibody at8

Anti-APP antibody (6E10) was purchased from Covance (Cat# SIG-39320-1000). Anti-beta amyloid was purchased from Abcam (Cat#2539). Anti-T. gondii antibody was purchased from US biologicals (Cat# T8075-01). BAG1 was a gift from Dr. Dubey. Normal mouse IgG (SC-2025) and normal rabbit IgG (SC-2027) were used as isotype controls to test the specificity of the 6E10 and BAG1 antibodies, respectively. Anti-phospho-Tau antibody (AT8) was purchased from Thermofisher (Cat# MN1020). Anti-NeuN was purchased from Cell Signaling (Cat# 12943S) or Abcam (ab104224). Anti-VGLUT2 (Cat#71555S), anti-GAPDH (Cat# 2118), and anti-Tau (Cat#4019) antibodies were purchased from Cell Signaling. Anti-NMDAR (Cat# ab17345), anti-VGLUT1 (Cat# ab134283), and anti-GAD67 (Cat# ab26116) antibodies were purchased from Abcam. Thioflavin S was purchased from (Sigma Aldrich) and neuro-tracer from Life Technologies.

Soluble fractions were thawed on ice and mixed with 4× NativePAGE sample buffer comprising 50 mM Bis-Tris, 50 mM NaCl, 10% (w/v) glycerol, and 0.001% Ponceau S, pH 7.2. Ten-microgram samples per well were loaded onto NativePAGE Novex 3–12% BisTris gels (ThermoFisher). The proteins were fractionated for 90–115 min at 150 V using an XCell SureLock Mini-Cell system (ThermoFisher, Carlsbad, CA). The fractionated proteins then were transferred to PVDF membranes for 1 h at 25 V using the XCell II™ Blot Module (ThermoFisher). After the transfer, the membranes were incubated in 20 mL of 8% acetic acid for 15 min to fix the proteins. The membranes were probed with anti-human tau antibody HT7 (ThermoFisher) or anti-phospho-tau antibody AT8 (ThermoFisher). The intensity of the protein bands was quantified densitometrically using Image J [31 ].