To begin, take the fixed transwell with lipofuscin-loaded RPE cultures. After washing the cultures with PBS five times, leave a small amount of PBS in the apical chamber. Place the transwell upside down under a dissecting microscope.
Using a razor blade, cut the semi-porous membrane from the transwell by applying the cutting force at the junction of the membrane and the transwell. Once the transwell membrane is cut, place it onto a microscope slide using forceps. Wipe away excess PBS with a task wipe while avoiding touching the cells.
Add mounting medium followed by a cover slip. Ensure to keep track of which side of the transwell is right side up. Next, detect the antigen of interest using a fluorophore, with peak excitation of around 488 nanometers and emission detection at 500 to 530 nanometers.
Set up a separate channel for autofluorescence detection, with 405 nanometers excitation and 585 to 635 nanometers emission. Autofluorescent granules induced by OxOS showed more accumulation after 20 feedings compared to five feedings. The ratiometric imaging helped to decipher UAM autofluorescence from LC3 labeled with Alexa 488.
The red channel contains only undigestible autofluorescence material and helped to separate the LC3 signal from the green channel.
