Mowiol solution

Retinal sections were permeabilized in 0.05% (v/v) TritonX-100 for 1 h and then were blocked with 10% (v/v) donkey serum (Sigma-Aldrich, St. Louis, MO, USA) for 1 h. Retinal sections were incubated with primary antibodies (Table 3) at 4 °C overnight in a humidity chamber. After washing with PBS × 1 for 3 times, retinal sections were then incubated for 1 h at room temperature with fluorescent secondary antibodies (Table 3). Some sections were continually incubated in TUNEL mixed solution for another 1 h. After washing with PBS × 1 for 3 times, sections were counter-stained with Hoechst 33258 and mounted in 10% (v/v) Mowiol^® solution (Polysciences, Eppelheim, Germany).
Retina whole mounts were permeabilized 0.2% (v/v) TritonX-100 for 30 min. The retina whole mounts were incubated in conical small wells and transferred with 3 mL pipette. The staining steps were the same as those of immunoistochemistry section. Finally, the retinal whole mounts were mounted on microscope slides and covered with thinner and smaller cover slides in 10% (v/v) Mowiol^® solution. Images of fluorescent sections and whole mounts were obtained using an epifluorescence Olympus BX60 microscope connected to a Hamamatsu C11440 digital camera.

Semithin (1 μm thick) sections were used for the analysis of chromatin condensation and apoptotic changes of nuclei. After removal of Araldite with 1% sodium ethoxide in absolute ethanol (40 min, 37°C), skin sections were rehydrated and stained with propidium iodide for 10 min. After rinsing in distilled water, the sections were mounted in Mowiol solution (Polysciences, Eppelheim, Germany) in order to preserve fluorescence signal and examined with a Carl Zeiss LSM 510 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany).