The role of the Wnt signaling pathways in retinal degeneration

Optic nerve crush (ONC) injury was performed at age 2 months. Mice were anesthetized using a ketamine/xylazine cocktail injected intraperitoneally and eyes were locally anesthetized using 0.5% proparacaine hydrochloride. Mice of either sex were randomly assigned into Wnt or saline control groups, and were intravitreally injected with 20 ng of recombinant Wnt3a or the equivalent volume of PBS (saline) into the same eye that had the optic nerve crush injury. The intravitreal injections of Wnt3a or saline control were made immediately following axonal injury. My biggest role was in analysis of the retinas through immunohistochemistry (IHC) followed by Retinal Ganglion Cell (RGC) counting. Cryoembedded eyes were sectioned in 10 μm sections, mounted onto slides and immunostained using anti-β-gal, anti-pan Brn3, anti-RPBMS, or anti-IBA1. Retina sections were counterstained using DAPI to label the nuclei, and imaged using a Zeiss Axiovert 200 fluorescent microscope. Control immunostaining using an irrelevant antibody or omitting the primary antibody was used to confirm antibody specificity. For RGC counts, Brn3-positive or RPBMS-positive and DAPI-positive cells within the GCL were counted.