FIGURE 1.

Retinal wholemount tissue processing and controls for autofluorescence. (A) Representative drawing of a left wholemount human retina. Punches (3.5 mm diameter) were taken from foveal (F) and the peri-foveal (PF) regions. The PF punch was taken midway between the foveal punch and the optic nerve head. Additional punches were also taken from superior (S), temporal (T), and inferior (I) quadrants in the mid-peripheral retina. Each mid-peripheral punch was taken approximately 4 disc-diameters from the optic nerve head. (B–F) Negative control section of neuroretinal wholemount with DAPI-only staining. Confocal microscope imaging at wavelength 405 nm (B), 543 nm (C), 488 nm (D), 405 and 543 nm (E), 543 and 488 nm (F). There was very low background fluorescence. No autofluorescence was visible. (G–I) Neuroretinal wholemount from an AD eye processed for immunofluorescence with anti-Aβ antibody (clone 6F/3D) and imaged at wavelengths 488 nm (G), 405 nm (H) and 563 nm (I). The immunofluorescence seen in panel (I) is specific for Aβ, as the secondary antibody was labeled with Cy3. Under excitation wavelengths of 405 nm (H) and 488 nm (G) the immunofluorescent cell seen in panel (I) does not display an autofluorescent signal from lipofuscin or melanopsin. Asterisk (*) in (G–I) indicates a landmark blood vessel.