Immunostaining, EdU staining, confocal imaging, and 3D reconstruction

Serial coronal brain sections (40 μm in thickness) through the entire forebrain were cut by a cryostat or a sliding microtome and were immunostained with the following antibodies: anti-GFP (1:200 or 500; goat; Rockland), anti-GFP (1:500; chicken; Aves Labs), anti-RFP (1:200 or 1,000; rabbit; Rockland; to detect tdTomato), anti-Nestin (1:500; chicken; Aves Labs), anti-goat/chicken Cy2, anti-rabbit Cy3, and anti-goat Cy5 (1:200 or 500; donkey; Jackson ImmunoResearch). EdU was visualized on cryosections in the detection solution (5 uM Sulfo-Cy3 azide [Lumiprobe], 0.1 M Tris pH 7.5, 4 mM copper sulfate, 100 mM sodium ascorbate) for 30 min after permeabilization in 0.5% Triton-X100 for 30 min. Consecutive sections covering individual clones were imaged using Zeiss LSM 710 confocal microscope (Carl Zeiss). For 3D reconstruction, optical stacks from the entire diencephalon were serially aligned along the rostro-caudal axis using Reconstruct 1.1.0 (J.C. Fiala, NIH), followed by import into Imaris (Bitplane) for further analysis.