## 3 Results
As a result, we observed that 2 mM, 1 mM, 500 uM, and 250 uM of fucoxanthin was colocalized with ThS-positive staining in the hippocampus and cortex of both APP/PS1 and 5\(\times\)FAD AD brain tissue when a water-based (PBS) washing solution was applied (Figures 3 and 4). Meanwhile, we confirmed that neither ThS nor fucoxanthin detected anything in wild-type mice (WT), where the AD phenotype is not observed (Figure S1 in the supplementary material). Our results indicate that fucoxanthin can detect A\(\beta\) aggregates in both APP/PS1 and 5\(\times\)FAD mice with amyloid pathology, suggesting that fucoxanthin has an amyloid plaque specificity while excluding the possibility for ThS-positive tau aggregates observed in 5\(\times\)FAD. Fucoxanthin Staining Using an Organic Solvent-Based Washing Detected ThS-Stained Amyloid Aggregates in AD Transgenic Mouse  
For reducing the nonspecific signal present in the brain sections during staining, the selection of washing buffer to eliminate fluorescent probes with lower binding affinity is an important consideration. Basically, the most frequently used buffer without destruction of cellular structure is phosphate-buffered saline (PBS). In this study, fucoxanthin from microalgae was extracted using ethanol, so this should take into account for the selection of buffer in the washing step. Thus, the A\(\beta\) staining ability of fucoxanthin needs to be evaluated by confirming its colocalization with ThS staining when using organic solvent-based washing solution in the fixed brain sections of AD transgenic mouse models. Similarto the water-based washing solution application, A\(\beta\) plaques in APP/PS1 and 5\(\times\)FAD brain tissues were stained with different concentrations of fucoxanthin (2 mM, 1 mM, 500 \(\upmu\)M, and 250 \(\upmu\)M) when an organic solvent-based (ethanol) washing solution was applied. These were colocalized with ThS staining (Figures 5 and 6). Taken together, these results suggest that fucoxanthin is a promising fluorescent imaging dye that can specifically detect amyloid aggregates in the AD transgenic mouse models regardless of the washing solution method. Figure 3: Fucoxanthin staining using the water-based washing method detected ThS-positive A\(\beta\) plaques in APP/PS1 brain tissue. The brains of 10–15-month-old APP/PS1 mice (\(n=4\)) were visualized for A\(\beta\) plaques with fucoxanthin (Fx) and ThS. The images of ThS and Fx are merged to confirm that they are colocalized. Scale bars = 200 \(\upmu\)m. The region of brain was separated with the white dash line on the images; upper region of dash line, cortex; under region of dash line, hippocampus. _Appl. Sci._**2021**, _11_, 5878  
Figure 4: Fucoxanthin staining using the water-based washing method detected ThS-positive A\(\beta\) plaques in 5\(\times\)FAD brain tissue. The brains of 10–15-month-old 5\(\times\)FAD mice (\(n=4\)) were visualized for A\(\beta\) plaques with fucoxanthin (Fx) and ThS. The images of ThS and Fx are merged to confirm that they are colocalized. Scale bars = 200 \(\upmu\)m. The region of brain was separated with the white dash line on the images; upper region of dash line, cortex; under region of dash line, hippocampus. Figure 5: Fucoxanthin staining using organic solvent-based washing method detected ThS-positive A\(\beta\) plaques in APP/PS1 brain tissue. The brains of 10–15-month-old APP/PS1 mice (\(n=4\)) were visualized for A\(\beta\) plaques with fucoxanthin (Fx) and ThS. The images of ThS and Fx are merged to confirm that they are colocalized. Scale bars = 200 \(\upmu\)m. The region of brain was separated with the white dash line on the images; upper region of dash line, cortex; under region of dash line, hippocampus.