Chicken anti gfp antibody

Whole mount immunofluorescence was performed as described in Ref.^{69 (link)}. Following FISH, a rabbit polyclonal anti-TH primary antibody^{22 (link)} was used at 1:500 dilution and detected with an anti-rabbit Alexa555-conjugated secondary antibody (2 µg/ml, Life Technologies A-21428). For double FIHC (Tg(top:dGFP)^w25 and Tg(7xtcf-Xla.siam:gfp)^ia4 embryos), a chicken anti-GFP antibody (5 µg/ml; Invitrogen) was combined with a polyclonal rabbit anti-TH antibody^{22 (link)}. Primary antibodies were detected with an anti-chicken Alexa488 antibody (2 µg/ml; Life Technologies A11039) and an anti-rabbit Alexa555 antibody (2 µg/ml, Life Technologies A21430). Following FISH for sox2 and sox3, primary mouse-anti-Sox2 antibody (2.5 µg/ml, Abcam, ab171380) was used and subsequently detected using an anti-mouse-Alexa633 (2 µg/ml, Life Technologies A21050) antibody. Following EdU detection, the Sox2 antibody was detected using an anti-mouse-Alexa555 secondary antibody (2 µg/ml, Life Technologies A21422).

Sections were rinsed by PBS and blocked by 2% Albumin Bovine – 0.3% Triton – PBS for 1 h. Then, slices were incubated with 1:500 chicken anti-GFP antibody (Invitrogen, A10262) overnight and followed by 1-h incubation with 1:500 Alexa Fluor 488 goat anti-chicken (Invitrogen, A11039) secondary antibody and DAPI. Slices were washed by PBS after incubation with antibodies. Finally, slices were mounted on slides and covered with mounting medium.

For all immunostainings, zebrafish embryos were fixed in 4% PFA, washed in 1xPBT 3 times for 5 minutes and digested in a Trypsin- 2.5% EDTA solution for 30 minutes (on ice). Subsequently, they were washed in PBT 4 times for 10 minutes. The first solution change was performed on ice. To avoid unspecific binding of the antibodies, the embryos were blocked in a solution containing 10% goat serum, 1%BSA and 0.8% Triton in PBT for minimum 1 hour at room temperature. Blocking solution was replaced by the primary antibody mix chicken anti-GFP antibody [Life technologies, diluted 1:200)] and either rabbit anti-rx2 [Charles River (diluted 1:200)], rabbit anti-PH3 [Millipore, (diluted 1:400)] or mouse anti-PCNA antibody [Millipore (diluted 1:250)] and DAPI (diluted 1:1000) in the following solution: 1% goat serum, 1% BSA and 0.8% Triton. Embryos were incubated at 4°C for 2 overnights with primary antibodies. After washing in PBT for several hours, incubation with the secondary antibodies [anti- chicken antibody, Alexa 488 [Dianova (diluted 1:250)] and anti-rabbit antibody, Alexa 647 [Thermo Fischer (diluted1:250)] and DAPI, diluted 1:1000 in 1% goat serum, 1% BSA and 0.8% Triton solution was carried out for 2 overnights.

After overnight fasting, mice were anesthetized and transcardially perfused with 0.9% saline with heparin followed by fixative (4% paraformaldehyde, 15% picric acid, 0.1% glutaraldehyde in PBS). Brains were collected, post fixed overnight, and coronal sections were taken at every 50 μm.
Sections were washed and incubated with the rabbit anti-cfos antibody (Santacruz, 1:2000), and the chicken anti-GFP antibody (Life Technologies Corporation, 1:5000) in PB containing 4% normal goat serum, 0.1% glycine, and 0.2% Triton X-100 for 24 h at room temperature. After several washes with PB, sections were incubated in the secondary antibodies (biotinylated goat anti-rabbit immunoglobulin G [IgG]; 1:250 in PB; Vector Laboratories and goat antichicken Alexa-fluor 488; 1:200 in PB; Life Technologies) for 2 h at room temperature, then rinsed in PB five times, 10 min each time. Sections were then mounted with VectaShield antifade (Vector Laboratories). Fluorescent images of five to seven brain sections were captured with confocal microscope and analyzed by imaging Software (Image J).