Rabbit anti βiii tubulin

Optic nerve sections or whole retinas were washed in PBS (3 × 5 min) and then incubated in blocking solution (5% donkey serum, 0.5% Triton X-100, and 1% bovine serum albumin in PBS) for 1 h at room temperature (RT), followed by incubation in primary antibodies either overnight (optic nerves) or for 3–5 d (retinas), always at 4°C. The primary antibodies used were: rabbit anti-GFAP (1:2,000; Abcam), mouse anti-SMI32 (1:400; Covance), rabbit anti-S100β (1:200; Abcam), mouse anti-vimentin (1:100; Abcam), rabbit anti–βIII-tubulin (1:200; Cell Signaling Technology), mouse anti-GFAP (1:400; Sigma-Aldrich), mouse anti-pSTAT3 (1:100; Cell Signaling Technology), and mouse anti-STAT3 (1:2,000; Cell Signaling Technology). The next day, tissue were washed in PBS (3 × 5 min) and incubated with secondary antibodies conjugated to rhodamine (1:200; donkey anti–rabbit; Jackson ImmunoResearch Laboratories, Inc.) or FITC (1:400; donkey anti–mouse; Jackson ImmunoResearch Laboratories, Inc.) for 2 h (optic nerves) or 3 d (retinas) at RT. Optic nerve sections were washed in PBS (3 × 5 min) and mounted in ProLong Gold Antifade medium (Thermo Fisher Scientific). Retinas were incubated with the nuclear dye DAPI for 20 min, washed in PBS (3 × 5 min), and mounted in Vectashield (Vector Laboratories).

Whole-mounted retinas were incubated in primary antibodies for 3 to 4 days at 4°C. The primary antibodies used were: rabbit anti-β-III tubulin (1:200; Cell Signaling Technology, Danvers, MA, USA), mouse anti-Brn3a (1:200, Nippon Chemi-Con, Tokyo, Japan). Secondary antibodies were FITC-conjugated donkey anti-rabbit IgG, and rhodamine-conjugated donkey anti-mouse IgG (both from Jackson Immunoresearch Laboratories, Inc., West Grove, PA, USA). Retinas were counterstained with the nuclear dye DAPI (Life Technologies, Grand Island, NY, USA), and mounted in Vectashield.