Goat anti gfp

Larval fish at 4 dpf, 5 dpf and 6 dpf were fixed overnight at 4°C in 4% PFA, rinsed three times in 1X PBS, incubated in 100% Methanol at −20°C for 1 hour and then rehydrated step-wise into 1X PBS at room temperature. Larvae were then incubated in 100% acetone at −20°C for 11 minutes, rinsed three times in 1X PBS, then digested at room temperature in 10 μg/mL Proteinase K for 45 min. (4 dpf), 55 min. (5 dpf) or for 65 min. (6 dpf). Larvae were then rinsed three times in 1X PBS, fixed for 10 minutes in 4% PFA at room temperature, rinsed 3 times in 1X PBS and then incubated in 5% Donkey serum block diluted in 1X PBS-tween-20, supplemented with 1% DMSO (PBTD), for 3 hours. Embryos were then incubated in either rabbit anti-GFP 1:500 (Life Technologies, A-11122), goat anti-GFP 1:500 (Abcam, ab6673), mouse anti-HuC/D (Hu) 1:200 (Invitrogen), mouse anti-Acetylated tubulin 1:1000 (Sigma, T6793) or rabbit anti-5HT 1:1000 (Immunostar) overnight at 4°C. Embryos were then washed out of primary antibody in 1X PBS-tween-20, then incubated at room temperature in 1:700 secondary antibodies Invitrogen Alexa Fluor Donkey anti-Rabbit 488, anti-Rabbit 647, anti-Goat 488 or Donkey anti-Mouse 594 for 3 hours at room temperature. Embryos were rinsed in 1X PBST and imaged in 75% glycerol/1X PBS on a Zeiss 710 2-photon confocal microscope (Beckman Imaging Center, Caltech).

Anesthetized mice were transcardially perfused with PBS followed by 10% Formalin. Skulls containing whole brains where kept overnight at RT in 10% Formalin. Brains were removed the following day and incubated in 10% Formalin overnight at 4C and then dehydrated in 30% sucrose for 3 days. Brains were hemisected sagittally, frozen in OCT, and cut in 12 µm sagittal sections using a Leica CM1860 UV cryostat (Leica Biosystems, Wetzlar, Germany). Primary antibodies include: Goat anti-GFP (Abcam, Cambridge, UK), Rabbit anti-CD8α (Sino Biological Inc., Beijing, China) and anti-CD4-PE (GK1.5, BioLegend). Secondary antibodies against goat (bovine anti-goat AF488) and rabbit (donkey anti-rabbit AF647) were purchased from Jackson ImmunoResearch (West Grove, PA). Images were acquired using a Leica DM4000 B LED microscope (Leica Biosystems, Wetzlar, Germany).