Dylight 633 conjugated streptavidin

The slices containing biocytin-filled neurons were fixed overnight (4% paraformaldehyde in 0.1 M PBS; pH 7.4; 4°C), and subsequently placed in PBS at 4°C. To visualize biocytin, the slices were incubated for 2 h at room temperature in DyLight 633 conjugated streptavidin (21844, Thermo Fischer Scientific). Then, the slices were washed in PBS (3 x 10 min), permeated with 1% Bovine Serum Albumin (BSA), 5% Donkey Serum (NGS), 0.1% Fish Gelatin, and 0.2% Triton x-100 for 1 h and incubated in sheep anti-Chx10 antibody 1:100 (AB9016, Chemicon) at room temperature for 48 hrs. Slices were washed in PBS (3 x 10 min) and incubated for 2 h at room temperature with rabbit anti-sheep Dylight 488 1:400 (SA5-10054, Thermo Fischer Scientific) followed by a final wash in PBS (3 x 10 min). Slices were then placed on slides within tissue spacers and coverslipped with a Vectashield mounting medium (Vector labs). Sections were scanned using a laser scanning confocal microscope (Leica True Confocal System SP8) in stacks of 10 optical sections across approximately 50 μm at 20x magnification. Images were condensed into maximum projections using the Leica collection software and brightness and contrast were adjusted in ImageJ.