Sp8 inverted confocal fluorescence microscope

Immunofluorescence was performed to reveal the in vitro distribution of Aβ-o. ~1.2×10⁵ SH-SY5Y cells/well were grown on an 8-well chamber slide (μ-Slide, Ibidi) overnight. SH-SY5Y cells were incubated with the samples of Aβ-o with or without GQDs for 3 h. After gently washed with PBS, the cells were fixed by 4% of paraformaldehyde for 15 min. A11 antibody (1:400, 70 μL) was then incubated with the cells at 4 °C overnight. After removing excess primary antibody and rinsing thoroughly with PBS, donkey anti-rabbit Alex 594 secondary antibody (1:500, 70 μL) was incubated with the cells at room temperature for 2 h. Then the cells were washed with PBS and further stained with Hoechst 33342 (1:1000) for 5 min. The chamber slide was transferred to a Leica SP8 inverted confocal fluorescence microscope, and the cells were observed and imaged using a 63×/1.40 numerical aperture oil immersion objective.

1.4×10⁵ SH-SY5Y cells/well were seeded onto 8-well chamber slide (μ-Slide, Ibidi) and cultured overnight in a humidified, 37 °C, 5% CO₂ incubator. Aβ oligomers (20 μM) or ultrasmall MoS₂ QDs (10 and 100 μM) were incubated with cells for 3 h. Cell culture media were used as negative control. Cells were gently washed twice with phosphate-buffered saline (PBS), and 4% of paraformaldehyde was added to fix the cells at room temperature for 15 min. After that, immunofluorescent staining was performed to reveal the distribution and organization of Aβ-o and actin filaments. Primary rabbit anti-oligomer polyclonal antibody (Invitrogen, 1:400) was incubated with the cells at 4 °C overnight, then donkey anti-rabbit Alex 594 secondary antibody (Abcam, 1:500) was used to conjugate with the primary antibody at room temperature for 2 h. Actin filaments were labelled with phalloidin-iFluor 488 (Abcam, 1:1000) at the same time. Then the cells were washed with PBS and further stained by Hoechst 33342 (Sigma, 2 μg/mL) for 5 min. After washing twice with PBS, the cells were observed with a Leica SP8 inverted confocal fluorescence microscope.