Figure 1. Histelide method.

A. 1 – Formalin-fixed, paraffin-embedded tissue was cut, mounted on glass slides, and deparaffinized; 2 – Slides were washed with TBST and incubated overnight in the blocking solution; 3 – Incubated with primary antibody in blocking solution, followed by washes with blocking solution; 4 - Incubated with secondary antibody in blocking solution, then washed with TBST and DEA solution; 5 - Incubated in the PNPP solution; 100 µL of the solution collected at predetermined intervals, and absorbance read at 405 nm; 6 – Slides were washed with TBST and NTM, incubated in BCIP solution for 30 min, and then washed in PBS and water, dried, coverslipped, and examined by light microscopy. B. PNPP₁₀₀ was determined by averaging absorbance values at 80, 100, and 120 min of incubation if all three values were ≥ 0.5. If absorbance values at these time points were < 0.5, then PNPP₁₀₀ was calculated by averaging absorbance at 200, 220, and 240 min time points and extrapolating to 100 min. C. Signal Intensity for gray matter, SI_GM, was calculated as the difference between the gray matter unit intensity (gray matter signal per cm² of tissue) and background unit intensity; we assumed that any signal from pure white matter section for synaptophysin, PHF-tau, or Aβ was background immunoreactivity. Gray matter unit intensity was obtained by subtracting white matter signal from PNPP₁₀₀ for the entire section that was composed of gray and white matter (Step 2).