Amyloid Thioflavin-S staining and Tau immunohistochemistry

Because our attempt to perform immunohistochemistry for Tau using snap-frozen brain sections failed, we looked for PFA-perfused-fixed brains from one of our partners of the INMiND consortium. Perfused-fixed brains from 18 m old animals were a generous gift from Dr Guadalupe Soria (IDIBAPS, Barcelona, Spain) and were used for Phospho-Tau immunohistochemistry and amyloid Thioflavin-S staining. PFA-perfused-fixed brains were cryoprotected in 30% sucrose, sectioned into 30 µm thick sagittal sections using a Leica frozen microtome and stored as free-floating brain sections at 4 °C in PBS/0.3% azide. Phospho-Tau and Thioflavin-S co-staining: sections were permeabilised by incubating with 0.3% Triton-X 100/PBS then endogenous peroxidase was quenched in 0.5% H2O2 for 30 min, and blocked in 2.5% normal horse serum (MP-7422, Vector Laboratory) followed by incubation with AT8, CP13 or PHF-1 primary antibody overnight at 4°C. Secondary Anti-Mouse IgG (MP-7422, Vector Laboratory) was applied for 30 min, followed by peroxidase substrate solution (SK-4105, Vector Laboratory) until desirable stain intensity developed. Sections were then incubated in 1% Thioflavin-S (T1892, SIGMA) solution for 7 min and washed with 70% Ethanol, before mounting with Fluoromount-G medium (Southern Biotech).

The entire hippocampus was imaged in each section using an EVOS FL Auto microscope (Life Technologies) with a ×20 objective, by area defined serial scanning (n = 3 per genotype). The image was processed using EVOS FL Auto Cell Imaging System and Adobe Photoshop CS6.