4.6.1. Fluorescence Microscopy Analysis—Acridine Orange/Ethidium Bromide (AO/EB) Dye

Visualization of the protective effects of various test samples (APO and APO-NPs) on PC12 cells was compared by subjecting them under AO/EB dye. In this double staining method, AO plays a significant role in cell-cycle studies as it can permeate through the cell and emit green fluorescence when bound to dsDNA by intercalating with the nucleic acid. In addition, being a metachromatic dye, acridine orange stains DNA green and RNA orange under appropriate conditions. However, EB imparts red/dark orange fluorescence when there is an altercation in cell membranes by intercalating inside the DNA [40]. Here, all the test samples were resuspended in 25 µL of a dye blend, formed by adding 100 µg/mL of each (AO/EB) dye in PBS (1 mL). Thereafter, 10 µL of the prepared sample was fixed on the slide with a coverslip for observation under a fluorescence microscope (Nikon Eclipse E 800).