A new protocol for live imaging of mammalian retina ex vivo by confocal laser scanning microscopy

This protocol describes a new procedure for the labelling of living retinas ex vivo, in order to image them by means of optical techniques, typically Confocal Laser Scanning Microscopy (CLSM). Several reports have addressed the immunohystochemical analysis of retina and of photoreceptors, mostly by inclusion or on previously fixed tissue (1), therefore with the exclusion of vital dyes. The retinal histoarchitecture has been evaluated by light microscopy of the inferior portion of the eye wall fixed and embedded in epoxy resin (2). The structure of isolated mammalian fixed retinas was also analysed by immunohystochemistry and CLSM (3,4). Our procedure maintains the physiological features of the tissue. Freshly enucleated bovine eyes are cut in half. The cornea, lens and vitreous are removed and discarded. The retina attached to the RPE is incubated into the eye semi-cup, in an appropriate reaction mixture, in dim light. A fluorescent dye (i.e. MitoTracker Deep Red 633) was added. Then, eyes or detached retinas are mounted for imaging. No other protocols are currently available to label living retinas still attached to the eyecup. By this technique we showed that rod outer segments and mitochondria are stained by MT and JC-1, out of a whole living retina (5).