4.7. Immunohistochemistry

Immunohistochemistry was performed to assess the distribution of phosphorylated tau at Ser202/Thr305 in the brain of PS19 mice using the AT8 antibody. Serial coronal sections (30 µm) were cut from the brains of PS19 and WT mice at 2, 4, 6, 8 and 12 months of age on a cryostat (Leica CM 1950) at −20 °C. Sections were stored at −20 °C in cryoprotectant (30% v/v glycerol, 30% v/v ethyl glycol, 20% v/v 0.1 M phosphate buffer and 20% v/v H₂O) until staining.

Unless stated, all immunohistochemical steps were performed at room temperature. Between each step, triplicate washes in phosphate buffered saline (PBS) were performed on an orbital shaker. The sections containing the frontal cortex, dorsal striatum, dorsal hippocampus and entorhinal cortex were selected from all animals and processed at the same time. To remove any aldehyde radicals from fixation and endogenous peroxidase activity, the sections were respectively incubated in 0.1 M glycine in PBS for 10 min and then 1% v/v hydrogen peroxidase (H₂O₂) and 40% v/v methanol in PBS for 30 min. The sections were then blocked with 10% v/v normal goat serum in blocking buffer (1% wt/vol bovine serum albumin and 0.3% v/v triton in PBS) for 1 h. Overnight, all sections were incubated with mouse AT8 primary antibody (1:1000 in blocking buffer) at 4 °C. All sections were then incubated in a biotinylated link solution containing biotin-labelled affinity isolated goat anti-mouse antibody for 90 min, followed by a 60 min incubation with a streptavidin/HRP complex solution. Phosphorylated tau Ser202/Thr205 (AT8) immunoreactivity was visualised as a brown colour due to the reaction with 3,3’-diaminobenzidine and urea/H₂O₂ tablets that were dissolved in 5 mL PBS. Sections were mounted on gelatine slides. The dried slides were put into a methanol series ending with two xylene incubations for dehydration and cover slipped with DPX mounting media. To confirm the specificity of the AT8 antibody, the primary antibody step was omitted from one section as a negative control. All images were captured using a Nikon microscope (Ti2E) mounted on a digital camera and NIS-Elements digital microscopy software.