Retinal whole-mount. GFAP-labeled retinal area (GFAP-RA) and NF-200+RGC counting. A: division of the retina in concentric zones for study and areas of retina selected from each zone; B: photomicrograph illustrating the astrocyte-counting methodology, GFAP+ astrocytes (in green) and DAPI, a nuclear marker (in red); C: photomicrograph of one of the areas of the retina selected for GFAP-RA quantification; D: same area shown in C processed with the threshold tool included in the Metamorph Imaging System. In red, GFAP-labeled retina included in the measurements and processing; E: double immunostaining photographs used to correlate the effect of OHT in both GFAP+ astrocytes (in green) and NF-200+RGCs (in red); F: same area shown in E used for manual counting of NF-200+RGCs. Fluorescence microscopy (B,C,D); Confocal microscopy (E,F). GFAP, glial fibrillary acidic protein; NF-200, 200 kD neurofilament; OHT, ocular hypertension; RGCs, retinal ganglion cells.