*2.6. Immunohistochemistry*

Immunohistochemical staining of mouse retinas was performed according to a previously published protocol [34]. Briefly, mice were euthanized and eyes were immediately enucleated and fixed in 4% paraformaldehyde (PFA) solution for 15 min. Cornea and lenses were carefully removed and posterior cups were incubated in 15% sucrose solution in phosphate-bu ffered saline (PBS) overnight at 4 ◦C after washing briefly in PBS. Posterior cups were transferred to 30% sucrose in PBS for 3–4 h, then embedded in optimal cutting temperature (O.C.T) medium and immediately frozen on dry ice. The frozen samples were stored at −80 ◦C until further processing. The sections were thawed at room temperature for 4 h, washed in PBS for 5 min, and permeabilized with 0.25% Triton-X in PBS for 5 min at room temperature. Sections were blocked with 10% horse serum in 1% bovine serum albumin (BSA) for 2 h then incubated with primary antibody diluted in blocking solution (1:100 dilution) overnight at 4 ◦C. The antibodies used were rabbit anti-GFAP (Abcam, MA, USA), mouse mAB HIF-1 α antibody (Novus Biologicals, CO, USA), LXR-β polyclonal antibody (Invitrogen, IL, USA), rabbit anti-Vimentin (Cell Signaling Technology, MA, USA), and isolectin GS-IB4 Alexa Fluor 568 (Life Technologies, OR, USA). Sections were then washed and incubated in fluorescent-labeled secondary antibodies (goat anti-rabbit IgG Alexa Fluor 488, Life Technologies, OR, USA) for 1 h at room temperature, followed by washing and incubation with <sup>4</sup>,6-diamidino-2-phenylindole, dihydrochloride (DAPI) solution (Life Technologies, OR, USA) for 5 min at room temperature. Finally, sections were washed and mounted with anti-fade mounting medium (Vector Laboratories, CA, USA) for imaging. Image analysis was completed in a masked fashion using four images taken at defined positions and quantified using ImageJ software. The analysis was performed in a masked fashion by three separate observers, then averaged. To achieve unbiased results, positive and negative controls were included alongside experimental test and control groups. Fluorescent microscopy was performed by trained masked operators. To address selection bias in immunofluorescence, the entire areas of retinal cross-sections were imaged.
