Retinal Cell Death Quantification

Samples were fixed overnight in 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M Sorenson phosphate buffer at pH 7.3. For bright-field imaging, samples were washed in PBS and imaged using an Olympus SZX10 microscope. For light microscopy, samples were post-fixed in 1% osmium tetraoxide and dehydrated in gradient ascending series of ethanol concentrations prior to Epon 812 resin embedding overnight. 1 µm sections were prepared using a Leica EM UC6 microtome and glass knife, mounted on glass slides and stained with toluidine blue. Prepared sections were imaged by a Leica DMLB bright field illumination microscope and Leica DFC 480 camera. The number of dying cells in the ciliary marginal zone (CMZ) was quantified by recording pyknotic nuclei present in sections 5 µm apart surrounding the optic nerve. The area of the CMZ was measured in central retinal sections using the polygonal selection tool in imagej, morphology data was analysed using an ordinary one-way ANOVA. The area of the CMZ was determined according to the criteria in Raymond, et al.^{69 (link)}. For transmission electron microscopy (TEM), 0.1 µm sections were prepared using a Leica EM UC6 microtome and diamond knife, transferred to a support grid, contrasted with uranyl acetate and lead citrate, and analysed on a FEI-Tecnai 12 BioTwin transmission electron microscope (FEI Electron Optics).