Animals were anaesthetized with a ketamine (100 mg/kg)/xylazine (20 mg/kg) cocktail, and perfused with ice-cold PBS followed by ice-cold 4% paraformaldehyde at approximately 70% cardiac output. Dissected brains were post-fixed overnight in 4% paraformaldehyde at 4 °C, and cryoprotected in 30% sucrose. For retinal immunohistochemistry, whole eyeballs were dissected and placed in ice-cold 4% paraformaldehyde for 10 minutes, and then washed in dPBS before dissecting the retina away from the rest of the eyeball and post-fixing in 4% paraformaldehyde overnight a 4 °C. Both brains and whole retinas were embedded in O.C.T. compound (Tissue-Tek) and 10 μm tissue sections were prepared with a Leica cryostat. The following antibodies were used: 1:5000 rabbit anti-GFAP (DAKO, Z0334), 1:500 rat anti-GFAP (Invitrogen, clone 2.2B10), rabbit anti-AQP4 (Sigma, HPA014784), 1:500 rabbit anti-RBPMS (PhosphoSolutions, 1830-RBPMS), 1:500 mouse anti-CD68 (AbD Serotec, clone 514H12), 1:500–1500 rabbit anti-hC3D (DAKO, A0063). Primary antibodies were visualized with appropriate secondary antibodies conjugated with Alexa fluorophore (Invitrogen).
