Cx22led brightfield microscope

Sections were rinsed 5 × 10 min in 0.1 M phosphate-buffered saline (PBS), were treated with 0.3% hydrogen peroxide in dH₂O for 30 min, and were incubated at 4 °C in primary antibody solution: 1:1000, goat anti-DCX (Santa Cruz Biotechnology, Santa Cruz, CA, USA) with 0.04% Triton-X in PBS, and 3% normal rabbit serum for 24 h. Sections were then rinsed 5 × 10 min in 0.1 M PBS and were transferred to a secondary antibody solution with 1:500, rabbit anti-goat (Vector Laboratories, Burlington, ON, Canada) in 0.1 M PBS for 24 h at 4 °C. Then, sections were washed 5 × 10 min in 0.1 M PBS and were incubated in ABC complex (ABC Elite Kit; 1:1000; Vector) for 4 h. Sections were then washed in 0.175 M sodium acetate buffer 2 × 2 min. Finally, sections were developed using diaminobenzidine in the presence of nickel (DAB Peroxidase Substrate Kit, Vector), mounted on slides, and dried. Sections were then dehydrated and coverslipped with Permount (Fisher Scientific).
DCX-expressing cells were quantified in 3 dorsal sections (−2.76 to −4.68 mm below bregma) and 3 ventral sections (−5.52 to −6.60 mm below bregma) using the ×40 objective using an Olympus CX22LED brightfield microscope. Areas of these sections were quantified using ImageJ (NIH, Bethesda, MD, USA) and were used for density calculations (number of cells per mm²).