Multi wavelength cell scoring module of metamorph software

To evaluate whether E-LPs affected RGC survival, retrograde labeling of RGCs with a retrograde fluorescent tracer Fluoro-gold (FG) was performed in a manner similar to that described previously.²³ (link)^,25 Briefly, rats were anesthetized with 5% isoflurane, then maintained in 2.5% isoflurane. Skin of the head was incised in the midline to expose the skull and sutured (sagittal, coronal, and lambdoid sutures). The incisions were subjected to bilateral 2 mm diameter craniotomies at 0.5 mm posterolateral to the sagittal and lambdoid sutures. Superior colliculi were carefully exposed by removal of the cerebral content. A small piece of sterile sponge (gelform; Pfizer, Pearl River, NY, USA), presoaked in 10 µl of 4% FG solution, was left on the surface of the superior colliculus. After the surgery, the rats were kept warm and allowed to recover. Seven days after the surgery, the rats were euthanized and the eyes were enucleated and fixed with SuperFix (Kurabo, Osaka, Japan) for 2 hours at 4°C. Retinae were removed from the sclerae and divided into four quadrants (superior, inferior, nasal, and temporal) and mounted on slides. Fluorescence images of each quadrant at 2.0 mm from the optic nerve head were acquired and analyzed by counting the number of FG-labeled RGCs using the Multi Wavelength Cell Scoring Module of MetaMorph software (Molecular Devices) to avoid measurement bias.