The specimens were washed with 0.1 M PB and then incubated with
20% skim milk (Dainihon–Seiyaku, Osaka, Japan) in 0.1 M PB containing
0.005% saponin (0.1 M PB-saponin; Merck) for 10 minutes to block
nonspecific antibody binding. They were then incubated with primary
antibodies diluted in 5% skim milk in 0.1 M PB-saponin for 24 hours at
4°C. Antibodies and concentrations used in this study were as
follows: mouse monoclonal anti-β-galactosidase (β-gal, 1:1000;
Promega, Madison, WI), rabbit polyclonal anti-β-gal (1:5000;
Chemicon, Temecula, CA), mouse monoclonal anti-nestin (1:1000;
PharMingen, San Diego, CA), mouse monoclonal anti-microtubule
associated protein (MAP) 2ab (1:100; Sigma, St. Louis, MO), mouse
monoclonal anti-MAP5 (1:1000; Chemicon), rabbit polyclonal anti-glial
fibrillary acidic protein (GFAP; 1:1000; Chemicon), rabbit anti-myelin
basic protein (MBP; 1:500; UltraClone, Wellow, UK), mouse monoclonal
anti-HPC-1 (1:1000; Sigma), mouse monoclonal anti-calbindin (1:500;
Sigma), and rabbit anti-rhodopsin (1:1000; LSL, Tokyo, Japan).
After the reaction with primary antibodies, the specimens were washed
with 0.1 M PB-saponin and incubated with secondary antibodies diluted
in 5% skim milk in 0.1 M PB-saponin for 90 minutes. Antibodies and
concentrations used in this study were as follows: fluorescein
isothiocyanate (FITC)-conjugated sheep anti-mouse immunoglobulin
(1:100; Amersham, Buckinghamshire, UK), FITC-conjugated donkey
anti-rabbit immunoglobulin (1:100; Amersham), Cy5-conjugated goat
anti-mouse IgG (1:200; Amersham), and Cy5-conjugated donkey anti-rabbit
IgG (1:200; Amersham).
Sections were then washed with 0.1 M PB, mounted with glycerol-PBS
(1:1) and observed with a laser-scanning confocal microscope (1024;
Bio-Rad, Hercules, CA).