Alexa fluor 488 goat anti rabbit

For immunostaining, sections were incubated in blocking buffer (5% normal goat serum and 0.1% Triton X-100 in PBS) for 1 h at room temperature. After washing with PBS three times, sections were incubated with the primary antibodies overnight at 4 °C and with the secondary antibodies for 4 h at room temperature. The primary antibodies used for staining were: rabbit anti-Iba1 antibody (1:200; Wako, Richmond, VA, USA), and cleaved caspase-3 (1:200; #9661S, Cell Signaling Technology, Danvers, MA). After incubation at 4 °C overnight, the cells were stained with Alexa Fluor® 488 Goat Anti-rabbit (#A11034) and 4′,6-diamidino-2-phenylindole (DAPI) (1:5000, (#D9542), Sigma-Aldrich) for 1 h. cleaved caspase-3 + cells within the GCL layer were counted for apoptotic cell staining. DAPI was used to mark the GCL layer and used to identify condensed nuclei. At least 5 retinal sections were counted per retina sample to give an overall landscape of cleaved caspase-3 positive cells per retina. Images were obtained using an Olympus Life Science IX83 Inverted Microscope.

In this experiment, autophagy was tested qualitatively by fluorescence microscopy as previously reported [14 (link)]. Cells (100,000 cells/well) were grown on glass cover-slips, and after reaching the suitable confluence, they were washed with HBSS and treated with cyclodextrin solutions (50 µM) or a chloroquine solution (100 µM) (positive control) overnight at 37 °C. After treatment, cells were washed, fixed (3.7 % PFA, 15 min, room temperature), and permeabilized (0.2 % Triton-X, 15 min, room temperature). After each step, cells were washed twice with HBSS. Then, the intracellular LC3B molecule was labelled with primary antibody (0.5 µg/mL, rabbit polyclonal antibody against LC3B; LC3B antibody kit for autophagy, Thermo Fisher Scientific, Waltham, MA, USA), (1 h, 37 °C), and fluorescently labelled secondary antibody (5 µg/mL, Alexa Fluor 488 goat anti-rabbit, Sigma-Aldrich Ltd., Budapest, Hungary) (1 h, 37 °C in dark). After labelling, cells were washed with HBSS three times, and the cell nuclei were marked with 283 nM DAPI. The final steps of the sample preparation and the fluorescence microscopy measurements were the same as described in Section 4.4.1.