A1sir confocal microscope

Samples were initially fixed with 4% paraformaldehyde for 10 mins at room temperature. Then, samples were processed with 0.1% Triton X for 5 mins, followed by 5% BSA blocking for 1 hr at room temperature. The slides were then transferred to the TUNEL (Biotium, 30074) working solution for 1 hr at 37 °C and then rinsed. To combine with Fluoro-Jade, the slides were then transferred to the Fluoro-Jade (Sigma, AG325) working solution for 10 mins and then rinsed, air dehydrated, xylene cleared. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI), and coverslips were placed. Immunostaining images were obtained with a fluorescence microscope (Nikon ECLIPSE Ti-S) or Nikon A1SiR Confocal Microscope.

All GCL DG ROI stacks, from Bregma −1.2 to −3.4mm, were obtained using a Nikon A1SiR confocal microscope (Melville, NY). Subtract background and thresholding (OTSU) were applied to all stacks using ImageJ software and ROIs were manually drawn around the granule cell layer (GCL) of the dentate gyrus (DG). Stacks of merged and split channels for CD68 and DCX were imported into Imaris software (ver. 8.3.1, Bitplane, Zurich, Switzerland) and the 3D-reconstructions for single (DCX, voxel=1.0; CD68, voxel=10) and colocalization (voxel = 1) channels were built from threshold values determined by ImageJ software (CD68, OTSU; DCX, OTSU). Surface area values of all 3D reconstructions were then divided by the volume from each ROI DG GCL z-stack obtained. To calculate the colocalization within the DCX and CD68 surface volumes, the colocalized surface volume value was divided by the CD68 and DCX surface volume values. In doing so, only the positive signal from CD68 and DCX found within the DG was examined, thereby removing the negative space from the colocalization calculation (Fig. 7A-F). For the co-culture experiment, 3D-reconstructions for the colocalization channel was built from threshold values determined by ImageJ software (CD68, OTSU; DCX, OTSU; Iba-1, Moments, Fig. 8).

Wild-type and tbx1 mutant embryos carrying the nkx2.5:Kaede transgene were photoconverted using a Nikon A1SiR Confocal Microscope (Nikon Instruments Inc.) and a 20X objective. 16 somite stage embryos were mounted in 0.9% low melting point agarose (Sigma-Aldrich) on 35 mm MatTek glass bottom Petri dishes (MatTek Corporation). Prior to photoconversion, embryos were imaged using a 488 laser to detect green fluorescence. Embryos were then exposed continually to UV light using the 405 mm laser for 3 min. Photoconverted animals were kept in a 28 C incubator until 32 hr post fertilization, anesthetized with 0.04% tricaine (Sigma-Aldrich), and imaged by confocal microscopy. The resulting z stack images were analyzed using FiJi/ImageJ software.

Tg(tbx1:mKate2P2Acre);Tg(ubi:HULK) double transgenic embryos were analyzed for GFP reporter signal using Nikon A1SiR Confocal Microscope (Nikon Instruments). Specifically, double transgenic animals were fixed overnight at 3 days post fertilization (dpf) with 4% PFA and processed by double immunohistochemistry for GFP and MF20. Similarly, Tg(tbx1:mKate2P2Acre); Tg(kdrl:CSY) double transgenic embryos were analyzed live for ZsYellow reporter fluorescence by confocal imaging using 405 nm blue diode and 514nm Argon lasers on a Zeiss LSM5 Pascal Laser Scanning Microscope (Carl Zeiss MicroImaging).