After a total of 7 days in vitro, cells were washed twice in
phosphate buffered saline (PBS; pH 7.3) and fixed with 4%
paraformaldehyde in PBS for 15 minutes at room temperature. Cells were
rinsed twice in PBS, permeabilized with 0.1% Triton X-100 for 5
minutes, and incubated in PBS containing 0.1% bovine serum albumin,
0.1% Tween 20, and 0.1% NaN
3 (buffer A) for 15
minutes. Different primary antibodies directed against various retinal
cell types were initially screened on frozen, paraformaldehyde-fixed
sections of rat and pig retina and cultured cells.
31 34 The following were chosen for this study: anti-neurofilament (NF)
68-kDa subunit monoclonal and anti-NF 200-kDa subunit polyclonal
antibody (Sigma–Aldrich; for RGCs and horizontal
cells
35 36 ); anti-neuron–specific enolase (NSE; for total
neurons
37 ) and protein gene product (PGP)9.5 (ubiquitin
decarboxylase; for RGCs and amacrine cells [ACs]
38 )
polyclonal antibodies (Chemicon, Temecula, CA); anti-protein kinase
Cα monoclonal antibody (Sigma–Aldrich; for rod bipolar cells[
BCs]
39 ); and anti-arrestin polyclonal antibody
(generous gift of Yvonne de Kozak, Institute National de la
Santé et de la Recherche Médicale [INSERM] Unité
450, Paris, France; for rod and cone photoreceptors[
PRs]
40 ). Coverslips were incubated with primary
antibodies (all diluted in buffer A and used at a final concentration
of 10 μg/ml) for 2 hours, washed six times with PBS, and exposed for
1 hour to goat anti-mouse IgG/Bodipy FL (10 μg/ml; for monoclonal
primary antibodies) or goat anti-rabbit IgG/Texas red (10 μg/ml; for
polyclonal primary antibodies; both from Molecular Probes, Portland,
OR). Nuclei of cultured cells were stained with
4,6-diaminodiphenyl-2-phenylindole (DAPI; 10 μg/ml, Sigma–Aldrich),
incubated together with fluorescent secondary antibodies. Finally,
coverslips and sections were washed thoroughly, mounted, and viewed
under a photomicroscope (Optiphot 2; Nikon, Tokyo, Japan), equipped
with Nomarski differential interference optics and epifluorescence
illumination.