For NR1 labeling, mice were perfused with ice-cold artificial cerebrospinal fluid (ACSF). Optic nerves were isolated and immersion fixed with 4% PFA for 2 hr at RT and prepared for later cryo-sectioning. Slide-mounted sections (12 μm) were air-dried at RT and then treated with 0.3% Triton X-100 and 5% horse serum for 1 hr. Primary antibodies for NR1 (1:250, Millipore Cat# MAB363, RRID: AB_94946) and MBP (1:300, rabbit, Dako) were incubated overnight at 4°C in the same solution. Secondary antibodies were incubated in 2% horse serum for 2 hr at RT.
For analysis of local inflammation and pathology, paraffin sections of perfusion-fixed tissues were used. Sections were treated with primary antibodies diluted in PBS/BSA (1% w/v BSA) overnight at 4°C. Dilutions were as follows: GFAP (1:200, mouse, Novocastra), Mac3 (1:400, rat, BD PharMingen), and APP (1:1000, Millipore Cat# MAB348 RRID: AB_94882). Biotinylated secondary antibodies were then incubated for 30 min at RT, and chromogenic staining was completed using HRP-DAB detection.
For analysis of local inflammation and pathology, paraffin sections of perfusion-fixed tissues were used. Sections were treated with primary antibodies diluted in PBS/BSA (1% w/v BSA) overnight at 4°C. Dilutions were as follows: GFAP (1:200, mouse, Novocastra), Mac3 (1:400, rat, BD PharMingen), and APP (1:1000, Millipore Cat# MAB348 RRID: AB_94882). Biotinylated secondary antibodies were then incubated for 30 min at RT, and chromogenic staining was completed using HRP-DAB detection.