Anti firefly luciferase

Allogeneic GRPs were isolated from the cortical region of the brain of a homozygous firefly luciferase transgenic mouse (FVB background, Jackson Laboratories, strain 008450) at the E13.5 stage, as described elsewhere (Mujtaba et al., 1999 (link)). Cells were maintained in serum-free DMEM-F12 medium (Invitrogen) supplemented with N2, B27, bovine serum albumin, and bFGF (Invitogen) as previously described (Lepore et al., 2006 (link)). GRPs were characterized using an immunocytochemical assay. In brief, cells were fixed in 4% paraformaldehyde (PFA) for 30 min. Nonspecific binding was blocked by incubating a solution of 10% donkey serum and 0.1% Triton X-100-PBS for two hours at room temperature. Cells were incubated with the appropriate dilutions of primary antibodies in blocking solution overnight at 4°C, then rinsed with PBS and incubated with corresponding secondary antibodies (Alexa Fluor-488 and Alexa Fluro-546, Invitrogen) in blocking buffer for one hour at room temperature. The culture was rinsed three times with PBS and images were obtained with a Zeiss AX10 fluorescence microscope. Primary antibodies used for these experiments were: anti-A2B5 (1:1000, Santa Cruz); anti-Olig1 (1:1000, EMD Milipore); and anti-firefly luciferase (1:3000, GeneTex).

At day 21 post-transplantation, mice were anesthetized and perfused intracardially with PBS followed by PBS-buffered 4% PFA. Brains were removed, postfixed overnight at 4°C, and cryopreserved in 30% sucrose. Serial coronal sections, 20 μm thick, were cut using a Thermo Scientific HM 550 Cryostat. For immunohistochemistry, nonspecific binding was blocked by incubating with a solution of 10% donkey serum and 0.1% Triton X-100-PBS for two hours at room temperature. Sections were then incubated overnight at 4°C with the appropriate dilution of primary antibody in blocking solution. The corresponding secondary antibodies (Alexa Fluor-488 and Alexa Fluro-546, Invitrogen) were added at a ratio of 1:200 in blocking solution for two hours at room temperature. Sections were then rinsed with 0.1 M PBS and placed on coverslips with aqueous non-fluorescing mounting medium (Immu-mount, Thermo Scientific). Images were obtained with a Zeiss AX10 fluorescence microscope. Primary antibodies used for these experiments were: anti-firefly luciferase (1:3000, GeneTex); anti-CD45 (1:500,Serotec); anti-CD11b (1:300, Biolegend); anti-GFAP (1:1000, Santa Cruz); anti-CD8 (1:500, Serotec); and anti-FoxP3 (1:1000, Abcam).