Fluorescence microscope 9 51

The brain tissue sections were stained with H&E following previous reports. The samples were subjected to observation under an optical microscope. H&E‐stained sections were visualized for morphological change of the cells of hippocampus in the brain. As for immuno‐histochemical staining, the brain tissue sections and cells were successively fixed, washed with PBS three times, and blocked with 0.5% Tritonx and 5% Bovine Serum Albumin (BSA) for 30 min at 37 °C. Followed by incubation of Aβ1‐42 antibody (1:1000), synaptophysin (1:1000), and GAP‐43 overnight at 4 °C, the samples then incubated by goat antirabbit immunoglobulin G (IgG) (H&L) FITC (1:1000) for 1 h at room temperature. After color developing and sealing, the sections were finally digitized using an Olympus fluorescence microscope (IX‐51).

The influence of nanocomposites treatment on HaCaT and A549 cells morphology and potential disruption of organelles were evaluated through fluorescence staining. Cells previously seeded in 12 well plates with density 1.5 × 10⁵ and 1.0 × 10⁵ cells/well, respectively were treated with chitosan–silver nanocomposites extracts (M52/VC) and co-incubated (24 h at 37 °C). Afterward, cells were washed with PBS (Ca²⁺, Mg²⁺) in triplicate and stained with organelle-specific dyes according to the manufacturer manuals.
Briefly, mitochondria were specifically stained with 100 μM solution of Mito Tracker® Green FM (Molecular Probes; Life Technologies; 470–495 nm excitation filter) in PBS with Ca²⁺, Mg²⁺. Fixation and permeabilization of cells, conducted before the F-actin visualization, were carried out by incubating the cells with 2% paraformaldehyde solution in PBS for 10 minutes at room temperature. Actin was stained with ActinGreen™ 488 ReadyProbes® Reagent (2 drops/1 mL of medium) (Molecular Probes; Life Technologies; 470–495 nm excitation filter) for 30 minutes at 37 °C. Cellular nuclei were stained with 2 μg mL^–1 Hoechst 33258 (Sigma-Aldrich; 530–550 nm excitation filter) solution in PBS for 10–20 minutes (for fixed and unfixed cells respectively) at 37 °C. An Olympus fluorescence microscope IX51 equipped with an XC10 camera was used for their visualization.

To study the effect of dcHGT NPs on metal‐induced Aβ fibrils, inhibition and disaggregation experiments were performed. In the inhibition study, 10 × 10⁻⁶, 25 × 10⁻⁶, and 100 × 10⁻⁶m Aβ1‐42 peptide were treated with 10 × 10⁻⁶, 25 × 10⁻⁶, and 100 × 10⁻⁶m Zn²⁺ or Cu²⁺ metal ions for 2 min at room temperature. Then 20 µL of dcHGT NPs (0.015, 0.0375, and 0.3 mg mL⁻¹) was introduced for 24 h at 37 °C. Similarly, a disaggregation experiment was performed by adding 20 µL of dcHGT NPs (0.015, 0.0375, and 0.3 mg mL⁻¹) into 10 × 10⁻⁶, 25 × 10⁻⁶, and 100 × 10⁻⁶m Aβ1‐42 fibrils for 24 h at 37 °C. The effects of Aβ fibril inhibition and disaggregation were confirmed by the TEM images and the ThT fluorescence assay. In order to trace the aggregation of Aβ1‐42, ThT (10 × 10⁻⁶m) was mixed into the solution and incubated for 1 h. ThT fluorescence images were acquired using an inverted Olympus fluorescence microscope (IX‐51).