Eyeball sections were deparaffinized and permeabilized using 0.05% Triton X-100. The samples was blocked by 3% bovine serum albumin and incubated with primary antibodies against caspase 3 (1:50), caspase 9 (1:100) (Proteintech Group, USA) overnight at 4 °C and washed with PBST. Sections were further incubated with anti-rabbit secondary antibody (1:500) (Jackson ImmunoResearch Inc., USA) followed by peroxidase staining with the peroxidase substrate 3,3-diaminobenzidine tetrahydrochloride. Sections were counterstained, mounted, and observed under a microscope.
For immunofluorescence staining of retinal cross sections, tissues were treated in 1% goat serum albumin/PBS for 1 h at room temperature before incubation with the primary antibody against PARP1 (1:200; Proteintech Group, USA) overnight at 4 °C. After several washes, sections were incubated with the secondary antibody Alexa Fluor 488 donkey anti-rabbit IgG antibody (1:100, Invitrogen, USA) at room temperature for 1 h and washed with PBS. The sections were counterstained with DAPI (Invitrogen, USA) in PBS. The immunostaining results were analyzed by Image-Pro plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).
For immunofluorescence staining of retinal cross sections, tissues were treated in 1% goat serum albumin/PBS for 1 h at room temperature before incubation with the primary antibody against PARP1 (1:200; Proteintech Group, USA) overnight at 4 °C. After several washes, sections were incubated with the secondary antibody Alexa Fluor 488 donkey anti-rabbit IgG antibody (1:100, Invitrogen, USA) at room temperature for 1 h and washed with PBS. The sections were counterstained with DAPI (Invitrogen, USA) in PBS. The immunostaining results were analyzed by Image-Pro plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).