Rabbit anti gfap

All animals were deeply anesthetized, perfused, and stained as previously described [7 (link)]. After perfusion, the brain was isolated, post-fixed overnight with 4% PFA, and sectioned with a vibratome at 50 μm intervals by embedding in 3% agarose in PBS (Leica, VT1000S). All sections through the hippocampus were collected in PBS containing 0.1% NaN₃ and stored at 4°C. Every twelfth section of brain tissue was permeabilized with 0.3% Triton X-100 in PBS, blocked for at least 2 hours at room temperature with 10% normal goat serum, and incubated with primary antibodies followed by secondary antibodies. For immunocytochemistry analysis, cells were fixed with 4% PFA for 20 minutes at room temperature, blocked with 10% normal goat serum in PBS containing 0.1% Tween20 for 1 hour, and incubated with primary then secondary antibodies. The primary antibodies used were: rabbit anti-GFAP (DAKO, 1:2000), rabbit anti-GFP (Proteintech, 1:500), rat anti-BrdU (Abcam, 1:500), mouse anti-PCNA (Santa Cruz, 1:200), mouse anti-Tuj1 (Sigma, 1:1000), rabbit anti-MAP2 (Millipore, 1:1000), rabbit anti-GFAP (DAKO, 1:1000), and mouse anti-O4 (R&D System, 1:1000). Fluorescent-conjugated secondary antibodies were used (Invitrogen, 1:1000). Biotinylated-conjugated anti-species IgG (Vector Laboratories, 1:500) were used for peroxidase/diaminobenzidine (DAB) staining in stereology analysis.

Immunofluorescence was performed as described previously (Stathopoulou et al., 2012 (link); Dimaki et al., 2013 (link); Iliou et al., 2013 (link); Champeris Tsaniras et al., 2018 (link)). Samples at day 1 and at day 15 were fixed in 4% paraformaldehyde for 20 min at 37°C. 0.5% Triton X detergent was then used to permeabilize the cells. Prior to staining with primary antibodies, samples were blocked with 3% (w/v) BSA in FBS. Samples from HEK and HeLa timepoints were stained with 1:1,000 anti-rabbit Ki67 (Zytomed, 2705) and 1:1,000 anti-mouse α-tubulin (Sigma, T8203) for 24 h, while samples from pRGCs were stained accordingly with 1:1,000 anti-mouse IgG1 Pax6 (DSHB), 1:1,000 anti-rabbit Sox2 (Abcam, ab97959) and 1:1,000 anti-rabbit GFAP (Dako, z0334), Ki67 (Zytomed, 2705) at day1 timepoint and at day15 timepoint were stained with 1:1,000 anti-rabbit GFAP (Dako, z0334) and anti-mouse α-tubulin. Samples were then stained with secondary fluorophore antibodies and Hoechst (Sigma). Cell morphology and proliferation was evaluated for the whole mounted samples inside imaging dishes (Ibidi, μ-Dish 35 mm) using a confocal microscope (Leica TCS SP5).