Primary antibody against gfap

For western blot analysis, whole retinas were used immediately or frozen at −70 °C until use. Retinal tissues were homogenized in a glass-Teflon Potter homogenizer in PRO-PREP^TM lysis buffer (iNtRoN Biotechnology, Inc., Seoul, Korea). Next, 10 µg of each sample was separated on a 10% polyacrylamide mini gel. After protein transfer, the membranes were blocked for 1 h at room temperature in Tris-buffered saline–Tween-20 solution [TBS-T; 10 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 0.1% Tween-20] containing 5% non-fat dry milk. After blocking, the membranes were incubated overnight at 4 °C with a primary antibody against GFAP (1:3000; Cell Signaling Technology, Inc., Danvers, MA, USA), Iba1 (1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and β-actin (1:10,000; Sigma, MO, USA). After three washes with TBS-T, the membranes were incubated for 1 h at room temperature with a peroxidase-conjugated goat anti-mouse IgG (1:3000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) or peroxidase-conjugated goat anti-rabbit IgG (1:3000; Cell Signaling Technology, Inc., Danvers, MA, USA) in TBS-T containing 5% non-fat dry milk. The signals were visualized by enhanced chemiluminescence and quantified using an LAS-3000 image analyzer (Fujifilm, Tokyo, Japan).

The brain sections (30 μm-thick) were soaked in 3% H₂O₂ with 20% methanol in PBS for 30 minutes to block endogenous peroxidase activity. After blocking in 4% milk solution for 1 hour, slices were incubated with primary antibody against GFAP (1:500; Cell Signaling Technology, MA, USA) overnight at 4 °C and then with biotinylated secondary antibodies for 1 hour. Staining was revealed by the avidin-biotin complex system (Vector Laboratories, CA, USA) and developed by using 0.5 mg/ml diaminobenzidine and 0.01% H₂O₂ in TBS to produce brown reaction product.

For western blot analyses, whole retinas were used immediately or frozen at −70 °C until use. Retinal tissues were homogenized in a glass-teflon Potter homogenizer in lysis buffer (PRO-PREPTM, iNtRoN Biotechnology, Seoul, Korea). Each sample (10 µg) was separated in a 10% polyacrylamide mini-gel. After protein transfer, membranes were blocked for 1 h at room temperature in Tris-buffered saline–Tween-20 solution [TBS-T; 10 mM Tris–HCl (pH 7.6), 150 mM NaCl, and 0.1% Tween-20] containing 5% non-fat dry milk. After blocking, membranes were incubated overnight at 4 °C with a primary antibody against GFAP (1:3000; Cell Signaling Technology, Danvers, MA, USA), Iba1 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), HIF-1α (1:1000; Santa Cruz Biotechnology), total-histone H3 (total-H3) (1:1000; Cell Signaling Technology), acetyl-H3 (1:1000; Cell Signaling Technology), or β-actin (1:10,000; Sigma Missouri, USA). After three washes with TBS-T, the membranes were incubated for 1 h at room temperature with a peroxidase-conjugated goat anti-mouse IgG (1:3000; Santa Cruz Biotechnology) or peroxidase-conjugated goat anti-rabbit IgG (1:3000; Cell Signaling Technology) in TBS-T containing 5% non-fat dry milk. Signals were visualized by enhanced chemiluminescence and quantified using a LAS-3000 image analyzer (Fujifilm, Tokyo, Japan).