Cells were fixed in 4% paraformaldehyde (Wako) for 1 hour and then immersed in 25% sucrose-PBS. After washing in 0.1 M phosphate buffer (PB), specimens were incubated for 1 hour with 20% skim milk (Dainihon-Seiyaku, Osaka, Japan) in 0.1 M PB, containing 0.005% saponin (0.1 M PB-saponin; Merck, Darmstadt, Germany), to block nonspecific antibody binding. Specimens were incubated for 24 hours at 4°C with primary antibody diluted in 5% skim milk in 0.1 M PB-saponin. Rabbit polyclonal anti-αA-crystallin (1:1000; Stressgen, Victoria, British Columbia, Canada) and anti-Pax6 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA) were used as the primary antibodies. The reactivity of the antibodies was confirmed by using rat lens as a positive control. After a wash in 0.1 M PB, specimens were incubated with the fluorescein-conjugated donkey anti-rabbit immunoglobulin (1:100; Amersham, Buckinghamshire, UK) secondary antibody diluted in 0.1 M PB-saponin with 5% skim milk for 1 hour at room temperature. After washes with 0.1 M PB, slides were mounted with glycerol-PBS (1:1) and observed by laser-scanning confocal microscope (Leica, Wetzlar, Germany).