Chicken anti gfp antibody

E17.5 embryos (2 wild type and 2 homozygotes) were removed from pregnant mice under deep ketamine/xylazine anesthesia. Cortices were papain-dissociated for 20 min at 32 °C and plated on 10 mm microcoverglass (Matsunami) coated with polyethyleneimine in a Nunc 12-well dish (Thermo Fisher). One culture from each animal was maintained at 37 °C in Neurobasal medium (Invitrogen) supplemented with B-27 (Invitrogen), humidified 5% CO₂ / 95% air. Neurons were transfected with pCMV-EGFP at DIV (days in vitro) 5 using Lipofectamine 3000 (Invitrogen) per manufacturer’s instructions. After 26 days in culture, neurons were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, then blocked with 1% normal goat serum and 2% bovine serum albumin. The neurons were then incubated overnight at 4°C with chicken anti-GFP antibody (1:500, Aves Labs). Goat anti-chicken AlexaFluor-conjugated secondary antibody (1:300, Invitrogen) was then applied for 2 h at room temperature. Immunofluorescent images were acquired by confocal laser-scanning microscope (FV1000-D; Olympus). Spine density was analyzed from 2 randomly selected neurons in each culture dish from individual animals by using the FilamentTracer module (v. 8.1.2, Bitplane) within Imaris software. Spine formation by WT and homozygote neurons were compared by unpaired t-tests.

Animals were sacrificed 4-6 weeks post-injection and perfused with 4% PFA. The brains were dissected out and fixed in 4% PFA for two additional hours at room temperature, rinsed with 1X PBS, and placed in 30% sucrose solution overnight at 4°C. To visualize viral expression and fiber placement, 60 μm sections were cut on a Leica CM1950 cryostat. To examine dLight expression in the NAc, 60 um sections were stained with chicken Anti-GFP antibody (Aves Labs #1020, 1:1000) overnight at 4°C, and with donkey anti-chicken Alexa 488 antibody (Jackson ImmunoResearch #703-545-155, 1:1000) overnight at 4°C. Images were acquired using a confocal microscope (Zeiss LSM 880).

eGFP expression in mouse kidneys was detected by immunofluorescence analysis, as described previously [11 ]. Two weeks following vector administration, the kidneys were collected and fixed in 3.8% para-formaldehyde for 1 h at 4 °C. After washing with PBS (3 times, 5 min each), the tissues were equilibrated with 30% sucrose in PBS overnight. Six µm cryosections were immunostained for eGFP using a chicken anti-GFP antibody (Aves Labs Inc. Tigard, Oregon). Biotinylated Lotus tetragonolobus agglutinin, which binds strongly to the brush border membrane of the renal PT cells and weakly to the intercalated cells of the CD, was used to distinguish PTs and CDs from other nephron segments. Nuclei were stained with 4′,6-diamidino-2-phenylindole.