Fluoroshied with dapi

The sections were washed with PBS 3 times for 5 min. After this, they were incubated in a 0.001% solution of FJ-C made in PBS for 10 min. The sections were then washed with PBS 3 times for 5 min, and they were blocked with 10% normal goat serum and 0.2% Triton X-100 in PBS at 25°C for 1 h. Next, they were incubated with primary antibodies (rabbit anti-β-amyloid, ab2539) diluted 1:500 in 1% normal goat serum and 0.2% Triton X-100 in PBS at 4°C for 18 h. After this, the sections were washed 3 times with PBS and incubated for 1 h at 25°C with Dylight 650 goat antirabbit (ab96898, Abcam), diluted 1:500 in PBS with 1% normal goat serum. Then, they were washed 3 times for 5 min with PBS, and autofluorescence was quenched with 50 mM NH₄Cl in PBS for 15 min. Finally, after washing the sections with PBS for 3 min, they were mounted onto the slides from PBS and then coverslipped with Fluoroshied™ with DAPI (Sigma).
When immunostaining was performed exclusively for Aβ, the same procedure was followed omitting the first washes and the FJ-C incubation.
Images were obtained on a Nikon Eclipse Ti-S microscope equipped with a Digital Sight digital camera and with a Leica SP8 confocal microscope equipped with a Leica DFC350FX digital camera.

Following the instructions described by Schmued et al. (2005) (link), the brain sections were mounted onto slides from distilled water and air-dried for 30 min on a slide warmer at 50°C. After this, they were first immersed in a basic alcohol solution consisting of 1% sodium hydroxide in 80% ethanol for 5 min, then 2 min in 70% ethanol and 2 min in distilled water; followed by 10 min in a solution of 0.06% potassium permanganate. They were rinsed in distilled water and stained for 10 min in a 0.0001% solution of FJ-C (AG325, Merck) made in 0.1% acetic acid. The proper dilution was accomplished by first making a 0.01% stock solution of the dye in distilled water and then adding 1 ml of the stock solution to 99 ml of 0.1% acetic acid vehicle. The working solution was used within 2 h of preparation. The slides were then rinsed through three changes of distilled water for 1 min per change. Excess water was drained onto a paper towel, and the slides were then air-dried on a slide warmer at 50°C for at least 5 min. The air-dried slides were then cleared in xylene for 1 min and then coverslipped with Fluoroshied™ with DAPI (Sigma).
Images were obtained on a Nikon Eclipse Ti-S microscope equipped with a Digital Sight digital camera and NIS-elements imaging software.

The brain sections, prepared as described earlier, were transferred to separate wells containing 500 μl of PBS in a 24-well plate. After 5 min, the sections were transferred to different wells containing PBS. This process was repeated one more time for a total of three PBS washes. After this, the sections were incubated in a 0.001% solution of FJ-C made in PBS for 10 min. The sections were then washed 3 times in PBS (5 min each), and autofluorescence was quenched with 50 mM NH₄Cl in PBS for 15 min. Finally, after three additional PBS washes (3 min each), they were mounted onto slides from PBS and then coverslipped with Fluoroshied™ with DAPI (Sigma). Images were obtained on a Nikon Eclipse Ti-S microscope equipped with a Digital Sight digital camera and NIS-elements imaging software. See Table 1 for details.