Laser confocal microscope

Cells cultured on glass coverslips were rinsed in PBS and fixed in 4% paraformaldehyde for 10 minutes. After washing three times in PBS, the cells were permeabilized with 0.2% Triton X-100 for 15 minutes. To block nonspecific binding, the coverslips were incubated with 3% BSA for 30 minutes at room temperature, probed with primary antibodies overnight at −4°C followed by FITC-conjugated secondary antibody for 1 h at room temperature. Finally, cells were counterstained with Hoechst 33,342 (10 μg/mL; Sigma, USA) for 30 min and visualized under a laser confocal microscope (Leica, Germany).
For staining F-actin, cells were washed with PBS and fixed in 3.7% formaldehyde for 10 minutes at room temperature. After permeabilizing with 0.1% Triton X-100 in PBS for 5 minutes, cells were incubated with rhodamine-conjugated phalloidin for 20 minutes at room temperature. The coverslips were washed and nuclei were stained with Hoechst 33,342 for 20 min. Cells were analyzed with a laser confocal microscope (Leica, Germany).

The anesthetized rats were transcardially perfused with 0.9% saline. The eyes were excavated and the anterior segments were removed. The eyecups were fixed in 4% PFA for 1 h and dehydrated in graded sucrose solutions (20–30%) overnight at 4°C. The eyecups were then embedded in optimal cutting temperature compound (Tissue-Tek; Ted Pella, Inc., Redding, CA, USA) and snap-frozen at −80°C until they were sectioned (10 μm). The frozen sections were stored in a −20°C freezer. The slices were fixed in 4% PFA for 30 min and washed three times with PBS. They were then treated with 0.5% Triton X-100 (Beyotime, Shanghai, China) for 15 min. The slices were blocked with 10% goat serum in PBS for 1 h at room temperature and then incubated with a primary antibody overnight at 4°C. Antibodies directed against the following proteins were used: p-P38 (1:100, Cat #4511, CST, Boston, MA, USA), p-JNK (1:100, Cat #4668, CST, Boston, MA, USA), or p-ERK1/2 (1:100, Cat #4370, CST, Boston, MA, USA). The slices were then washed three times with PBS, incubated with the corresponding secondary antibody (1:1000, Cat #A21428, Invitrogen, Carlsbad, CA, USA) for 1 h, and counterstained with 4′,6-diamidino-2-phenylindole (diluted 1:1000; Sigma-Aldrich, St. Louis, MO, USA). Finally, the slices were scanned with a laser confocal microscope (Leica Microsystems, Bensheim, Germany).