The flatmounted retinas were photographed with a scanning laser
confocal microscope (model LSM 410; Zeiss, Oberkochen, Germany) using a
green filter to detect FITC and a red filter for PI; the focus was in
the retinal GCL plane. The numbers of FITC-labeled c-Jun– and
caspase-positive cells, TdT-dUTP terminal nick-end labeling
(TUNEL)–positive cells and PI-stained cells were counted in six areas
(0.2 × 0.2 mm), 1 and 2 mm away from optic disc and every 30°
of the circle in each quadrant. Thus, data from 24 areas from one eye
were obtained. Regions with thick nerve fiber or blood vessels were
avoided, and a more central or peripheral area was chosen. Of the
PI-stained cells, white blood cells, which have fragmented nuclei, and
red blood cells, which have oval-shaped and spindle-shaped endothelial
cells, were excluded from cell counts. The cell count was done by two
examiners in a masked fashion. Data are expressed as the number of
cells per square millimeter at each time point, and results are
expressed as mean ± SEM.