2.6. Immunohistochemistry

After the novel object recognition test, mouse brains were isolated, and immunostaining for doublecortin (DCX) was performed using a free-floating technique. Mouse brains were removed, post-fixed using 4% paraformaldehyde overnight, and immersed in a 30% sucrose solution. Serial 30 µm coronal sections of the hippocampus were cut on a freezing microtome (Leica) and stored in cryoprotectant (35% ethylene glycol, 25% glycerol, 0.05 M PBS). Three brain sections per mouse were randomly selected for staining. To avoid selection bias and minimize topographical or dimensional differences between the pieces, we chose 1 per 10 section pieces. Three sections were analyzed per mouse—eight mice were dissected and total of 24 sections were examined for DCX cells.

The pre-floating slices were blocked with a protein block solution (GBI Labs). Subsequently, sections were stained with an antibody against DCX (sc-8066, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C. Sections were incubated with rabbit anti-goat IgG secondary antibody (Vector Laboratories) for 2 h at room temperature. The antibody against DCX was diluted to 1:200, while the secondary antibody was diluted to 1:300. Sections were then incubated with Vector ABC kit (Vector Laboratories). DCX-positive cells were visualized with 3,3'-diaminobenzidine (Vector Laboratories) and images were captured using a Leica DM5500B microscope (Leica). To quantify the total number of DCX-positive cells in the hippocampal dentate gyrus, cell counts were performed in at least three consecutive sections at a magnification of 40× using a counting grid, defining an area of interest to a width of 500 μm along the fissure by a blinded examiner.