Quantification of Retinal Ganglion Cells

The experiment was performed as described previously.²⁸ Briefly, whole-mount retinae were harvested at day 7 post-procedure, fixed in methanol, and immunostained with primary goat-anti-mouse Brn3a antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), which was recognized by Cy3-conjugated donkey anti-goat secondary antibody (Abcam, Cambridge, MA, USA). Samples were mounted with Vectashield mounting medium (Vector Laboratories) and examined by the AxioImager M1 epifluorescence deconvolution microscope with AxioVision 4.8 software (Carl Zeiss AG). For each retina, eight areas were randomly selected at a distance of 850 μm from the optical disc for data analysis.^{29 (link)} Digital images were analyzed to quantify the density of Brn-3a⁺ cells in each area (688 × 545 μm) using ImageJ software (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA). The percentage scores were obtained by normalizing to control condition defined as being 100%.^{24 (link)}