Mouse anti smi32

Fluorescent immunohistochemistry (IHC) was performed on 20-μm cryosectioned PFA-fixed brain tissue as described in refs. 67 (link) and 84 (link)–87 (link, link, link). Primary antibodies were diluted in blocking buffer and incubated on tissue sections overnight at 4 °C. The following antibodies and dilutions were used: mouse anti-Brn3a (diluted 1:125, Millipore), rabbit anti-RFP (diluted 1:500, Rockland), rabbit anti-Opn4 [diluted 1:2,000, Dr. C.K. Chen’s laboratory (67 (link))], mouse anti- SMI32 (diluted 1:1,000, Covance), rabbit anti-RBPMS (diluted 1:500, PhosphoSolutions), rabbit anti-GFP (diluted 1:250, Invitrogen), mouse anti-NeuN (diluted 1:200, Millipore), rabbit anti-GFAP (1:1,000, DAkoCytomation), rabbit anti-Iba1 (1:500, Wako), mouse anti-GAD67 (diluted 1:500, Millipore), rabbit anti-calbindin (diluted 1:2,500, Swant), rabbit anti-calretinin (diluted 1:2,000, Swant), mouse anti-synaptophysin (diluted 1:500, SySy), and goat anti-NPNT (diluted 1:40, R&D systems). A minimum of three animals (per genotype and per age) were compared in all IHC experiments. Please reference SI Appendix for more details.

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).