Ultrastructural Analysis of Photoreceptor Organelles

Mouse eyes fixed in 3% glutaraldehyde and 1% paraformaldehyde in 0.08 M sodium cacodylate buffer, pH 7.4, were sequentially en bloc stained with 1% osmium 1.5% potassium ferricyanide in 0.08M cacodylate buffer, 1% aqueous sodium thiocarbohydrazide, 1% aqueous osmium, 1% aqueous uranyl acetate followed by Walton’s lead aspartate (50 (link)). The samples were dehydrated using a series of increasing concentrations of ethanol (50, 70, 90, 3x 100%) followed by propylene oxide and infiltration in a mixture of propylene oxide and Durcupan ACM resin (1:1), before embedding in Durcupan ACM resin. Blocks cut from the embedded specimens were superglued onto aluminum pins prior to coating with gold palladium. Images were acquired in between sequential sectioning (100nm thick) of the block surface using a Gatan 3View system (Gatan Inc, Abingdon, UK) and a Zeiss Sigma VP field emission scanning electron microscope (Zeiss, Cambridge, UK). The images were re-aligned using the StackReg plugin (EPFL) in ImageJ (NIH) and modelling was performed using Amira 5.3.3 software (FEI). Measurements of photoreceptor inner segment ER and mitochondria of were made using 9 and 12 rods respectively from Reep6 KO and heterozygous mice in Amira and analyzed in Prism (Graphpad).