Abstract
Purpose :
Visualization of high resolution 3-dimensional cellular morphology in retinal flatmounts is limited. Various methods have been developed for clearing tissue to better visualize cells; however, these methods can be extremely time-consuming and can be deleterious to tissue integrity and/or are incompatible with fluorescent immunolabeling. Using a recently developed ultra-fast tissue clearing protocol using an alkaline solution (AKS), we have investigated the feasibility of imaging through murine retina tissue thickness to visualize retinal cell morphology in three dimensions.
Methods :
Adult wild type male mice were euthanized and eyes were removed. Eyes were drop fixed in 4% paraformaldehyde, rinsed in 1x PBS, retinas dissected from the eye cup, and 4 slits made to create a petal shape. Retinas were then incubated in -20oC methanol for 20 minutes and blocked in PBS containing 0.3% Triton-X 100, 0.2% bovine serum albumin, 10% goat serum for 1 hour before incubating in primary antibody. Following overnight incubation at 4oC of ionized calcium binding adaptor molecule 1 (IBA1) and glial fibrillary acidic protein (GFAP), retinas were rinsed in 1x PBS before secondary antibody incubation for 3 hours at room temperature. Retinas were then rinsed and placed in AKS solution (20% (vol/vol) DMSO, 40% (vol/vol) TDE, 20% (wt/vol) sorbitol, and 6% (wt/vol, equal to 0.5 M) Tris base dissolved in ddH2O) for 30 minutes on a shaker at room temperature. Retinas were placed in 35mm glass bottom dishes in AKS solution for confocal imaging using a Nikon AXR confocal. Retinal thickness and cell morphology was visualized using NIS-Elements analysis software.
Results :
The results showed that murine retinas were successfully cleared after 30 minutes of AKS treatment. Results also showed that AKS treatment did not alter fluorescent labeling of the retinal flatmounts. Using AKS allowed visualization of fluorescent labeling through the entire thickness of the retinas. Using high-resolution confocal microscopy, we were able to generate 3D images of our fluorescent labeling and visualize cell morphology.
Conclusions :
Retinal confocal imaging was done using AKS tissue clearing, showing that AKS allowed for deeper visibility of fluorescent markers in mouse retinal flatmounts. Using AKS tissue clearing on the retina can help better visualize cellular morphology of supporting cell types such as microglia and astrocytes throughout the entire retina.
This abstract was presented at the 2024 ARVO Imaging in the Eye Conference, held in Seattle, WA, May 4, 2024.