As described previously,
28 29 retinal ganglion cells
from P8 rat retina were purified by the immunopanning procedure.
Briefly, the retinal tissue was dissociated to single cells in Hanks’
balanced salt solution (HBSS) containing 15 U/ml papain and 70 U/ml
collagenase. The dissociated cells were incubated in a polypropylene
tube coated with an anti-rat macrophage monoclonal IgG (Chemicon,
Temecula, CA) diluted 1:50 to exclude macrophages and then incubated in
a tube coated with an anti-rat Thy 1.1 monoclonal IgG (Chemicon)
diluted 1:300. The tube was gently washed with PBS five times, and
adherent retinal ganglion cells were collected by centrifugation at
2000 rpm for 5 minutes. To determine purity, retinal ganglion cells
were labeled in a retrograde manner by injecting 1 mg/ml
1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine
(DiI)into the superior colliculi of anesthetized P5 rats. As
described previously,
29 during this immunopanning method,
approximately 85% of the collected cells were labeled by DiI. The
purified retinal ganglion cells were plated at a low density of
approximately 500 cells/cm
2 on 12-mm glass
coverslips coated with 50 μg/ml poly-
l-lysine and 10μ
g/ml laminin. The cells were cultured in Neurobasal medium (Life
Technologies, Rockville, MD) with 1 mM glutamine, 10 μg/ml
gentamicin, B27 supplement (Life Technologies), 40 ng/ml human
brain-derived neurotrophic factor (BDNF; Diaclone Research,
Besançon, France), 40 ng/ml rat ciliary neurotrophic factor
(CNTF; Diaclone Research) and 5 μM forskolin (Sigma, St. Louis, MO).
Cultures were maintained at 37°C in 5% CO
2 incubator. The immunohistochemical studies using anti-NGC antibody were
performed at 1, 3 and 7 days after incubation began.
Fluorescein-conjugated anti-rabbit IgG (Vector) was used as the second
antibody. Slides were examined under a confocal microscope.