Müller cells were cultured for 48 hours in
fibronectin-coated (5 μg/mL) glass chamber slides (NalgeNunc, Inc.,
Roskilde, Denmark), fixed in 4% paraformaldehyde in phosphate-buffered
saline (PBS; pH 7.2) for 10 minutes, and incubated for 3 hours with
primary antibodies diluted in 0.5% blocking reagent (Roche Molecular
Biochemicals, Lews, UK) in Tris-buffered saline (TBS; pH 7.5). These
included a monoclonal anti- CRALBP antibody (B2, a kind gift of John C.
Saari, University of Washington, Seattle, WA); goat polyclonal
anti-glutamine synthetase (clone C-20, Santa Cruz Biotech, Santa Cruz,
CA); monoclonal anti-EGF-R (clone 29.1, Sigma, Poole, UK); monoclonal
anti- α-SMA; clone 1A4, Sigma), and monoclonal anti-GFAP (clone 6F2;
Dako, Glostrup, Denmark). Mouse IgG isotypes matching those of the test
antibodies (Sigma) were used as the negative control. After incubation
with primary antibody, specimens were washed in TBS, followed by
incubation for 30 minutes with rabbit anti-mouse antibodies conjugated
with FITC or rhodamine (Santa Cruz Biotech). Slides were then washed
and counterstained with 4′,6′-diamino-2-phenylindole (DAPI) for 1
minute and mounted on glass slides (Vectashield mounting medium; Vector
Laboratories, Burlingame, CA). Fluorescent images were recorded using a
confocal microscope (LSM 510; Carl Zeiss, Oberkochen, Germany)
operating in multitrack mode for FITC, DAPI, and rhodamine-Cy3
fluorochromes.