The isolated retina was placed in a tube containing 1 ml of 25 mM
HEPES-Dulbecco’s modified Eagle’s medium (DMEM), pH 7.4. (Life
Technology, Grand Island, NY). Dissociated photoreceptors were then
obtained by vortexing with three 1- to 2-second pulses. After allowing
the pieces of retina to settle for ∼1 minute, the resulting
supernatant was collected, transferred to a new tube containing 1 ml of
HEPES-DMEM, and then plated on poly-l-lysine (5μ
g/ml)-coated coverslips. After 30 minutes at 4°C, a coverslip with
attached cells was rinsed with 0.1 M phosphate-buffered saline (PBS),
pH 7.4, and then fixed for 30 minutes with 4% paraformaldehyde in 0.1
M sodium cacodylate, pH 7.4. After rinsing with PBS, nonspecific
binding sites were blocked with 5% normal donkey serum in PBS, the
cells were incubated with both the monoclonal anti-rhodopsin antibody
(rho4D2) and the anti-FGFR1 antibody [Flg(C-15)] in PBS containing
0.5% bovine serum albumin, 0.1% Triton X-100, and 0.1% sodium azide
(PBTA) for 2 hours. The cells were then rinsed three times with PBTA,
incubated for 2 hours with a 1:200 dilution each of Cy3-labeled
anti-rabbit IgG and Cy2-labeled anti-mouse IgG (Jackson
Immunolaboratories, Inc., West Grove, PA) in PBTA, rinsed three times
with PBTA, mounted in 5% n-propyl gallate in glycerol, and
then examined by laser scanning confocal microscopy (MRC-1024; Bio-Rad
Laboratories, Hercules, CA).