Fig. 8
AM-NPs accelerate lysosomal clearance of fAβ. a–d BV2 microglia were incubated with NPs for 24 h and then co-incubated with Alexa Fluor 488-labeled fAβ for 2 h or 24 h. Lysosomes in treated live BV2 microglia were stained with 70 µM Lysotracker (red) for 30 min. Cells were then fixed using 4% PFA. a Representative confocal microscopy images showing fAβ-positive (green) lysosomes (red). White arrows show fAβ in lysosomes. Scale bar, 25 µm. b Quantitative measurement of the lysosomal degradation of intracellular Aβ at 2 h and 24 h in BV2 microglia pretreated with NPs. Data are presented as mean ± SEM; n = 3; ****P < 0.0001 for T12P5 versus fAβ, for Dunnett’s multiple comparisons shown on graph by two-way ANOVA. c, d BV2 microglia incubated with NPs for 24 h then co-incubated with Alexa Fluor 488-labeled fAβ for 2 h. Cells were fixed with 4% PFA, permeabilized using PBS-T, and then blocked using 2% goat blocking buffer. Cells were then incubated with anti-CD68 or LAMP-1 antibody overnight at 4 °C, then washed with PBS-T. Cells were incubated with secondary antibody Alex 488 or 954 for 1 h and nuclei were counterstained with Hoechst. c Orthogonal projection of confocal Z-stacks shows labeling of CD68 (green), Dil-labeled NPs (red), and nuclear stain Hoechst (blue). d Orthogonal projection of confocal Z-stacks shows labeling of LAMP-1 (white), Dil-labeled NPs (red), nuclear stain Hoechst (blue), and fAβ488 (green) in brightfield illuminated cells in the top panel, and co-labeling of nuclear stain Hoechst (blue), Dil-labeled NPs (red), and fAβ488 (green) in brightfield illuminated cells in the bottom panel. White arrows show intracellular fAβ. e, f BV2 microglia were incubated with NPs for 24 h and then co-incubated with fAβ for 30 min. e Confocal images of LC3 immunoreactivity (green) in BV2 microglia co-stained with Hoechst (blue). f Quantitative analysis of LC3 fluorescence intensity in BV2 microglia. Data are presented as mean ± SEM; n = 3; **P < 0.01 for T12P5 versus fAβ, for Dunnett’s multiple comparisons shown on graph by two-way ANOVA. Scale bar, 25 µm