A novel, rapid flat-mounting technique for visualizing antibody labeling in the retina

Complete analysis of retinal tissue is difficult because it consists of a thin neural tissue spread across the back of a hemispheric surface. Conventional sectioning in a plane parallel to a central axis of symmetry produces a large number of samples, each containing only a small amount of the tissue of interest. Consequently, quantitative comparison of any feature of interest typically uses a small fraction of the sections from each retina, because analysis of the entire collection of sections is too time consuming. Such a sampling process can lead to misleading or erroneous conclusions. We present a new method which allows complete analysis of the retina using a small number of samples produced by sectioning flattened retinas. This procedure is straightforward as illustrated using an antibody against proliferating cell nuclear antigen (PCNA) to locate dividing cells in the teleost fish retina. Immunocytochemical staining on flat-sectioned retinas was quantified using a computer-based image analysis system. When the cells of interest are randomly distributed, conventional sampling procedures can seriously under- or over-estimate their number. The new technique presented allows significantly more efficient examination and quantification of the entire retina as compared to conventional techniques.