Rabbit anti manf

Immunofluorescence staining was conducted as previously described Wu et al. (2013) (link). The eyes and RGCs from different groups were fixed with 4% paraformaldehyde (PFA) for 2 h and 20 min at room temperature, respectively. Then, the eyes were cut into 8-μm-thick sections after graded dehydration with 20 and 30% sucrose solution and then stored at -80°C until use. Sections and RGCs were incubated in 0.1% Triton X-100 and 3% (w/v) bovine serum albumin (BSA) (Sigma–Aldrich, St. Louis, MO, USA) for 30 min sequentially at room temperature to prevent non-specific background. Then, sections and RGCs were incubated with rabbit anti-MANF (1:200, Abcam, Cambridge, MA, USA) and anti-survivin (1:200, Abcam, Cambridge, MA, USA) antibody at 4°C overnight, followed by successive incubations with fluorescein-conjugated goat anti-rabbit secondary antibody (1:400, Molecular Probes, USA) and Hoechst staining. Negative controls were routinely prepared by incubating the cells and retinal sections in normal buffered serum instead of the primary antibody. The stained sections and cells were visualized and imaged using confocal microscopy (Leica SP8, Hamburg, Germany).

Immunofluorescence was detected on frozen 8 μm retinal sections and RGCs. Briefly, retinal and ON sections and the RGCs were incubated with rabbit anti-MANF (1:200, Abcam, Cambridge, MA) and mouse anti-glial fibrillary acidic protein (GFAP, 1:1000)/neuronal class III β-tubulin (TUJ1, 1:500)/ Thy1.1 (1:500, Abcam, Cambridge, MA) at 4°C overnight. Then, they were incubated with fluorescein-conjugated goat anti rabbit or mouse secondary antibody (1:400, Molecular Probes, USA) for 1 h at room temperature. In addition, slides were just incubated with fluorescein-conjugated goat anti rabbit or mouse IgG without primary antibodies to exclude non-specific binding. The slides were visualized and photographed by confocal microscopy (Leica SP8, Hamburg, Germany).