2.7.3. Fluorescence immunohistochemistry and analysis of BrdU and DCX

BrdU and DCX double fluorescence immunohistochemistry was performed on every 24th section, spaced at 480 μM, throughout the entirety of the hippocampus on a cohort of 12 animals. After removing the sections from − 20 °C and washing in phosphate buffer, sections were blocked for 30 min in 10% normal goat serum with 0.3% Triton-X then incubated in rabbit anti-DCX primary antibody (1:500; Cell Signaling 4604S) overnight at 4 °C. Sections were then washed and incubated in goat anti-rabbit Alexa Fluor 488 secondary antibody (1:250; #A-11034 Invitrogen) for 2 h at room temperature. Sections were then washed, fixed in 4% paraformaldehyde for 10 min at room temperature, incubated for 30 min in 2 N HCl at 37 °C, and blocked for 30 min prior to incubation overnight at 4 °C in primary antibody for BrdU (mouse anti-BrdU Roche 11170376001; 1:200). Sections were washed, incubated in goat anti-mouse Alexa Fluor 568 (1:250; #A-11004 Invitrogen) for 2 h at room temperature, and finally washed, mounted and coverslipped.

The estimated total number of BrdU+ cells in the granule cell layer was obtained by an operator blind to subject group. BrdU labeled nuclei were identified using a 100x oil objective on a Zeiss Axio Imager M2 (Carl Zeiss Microscopy GmbH; Jena, Germany), equipped with a motorized stage, and controlled by StereoInvestigator software (MicroBrightField; Williston, VT, USA). As BrdU labeling is rare, an optical fractionator with an exhaustive counting scheme including guard planes was used, as above. Tissue throughout the rostral-caudal extent of the hippocampus was analyzed for BrdU+ cells that were also positive for immature neuronal marker DCX. Double immunopositivity was confirmed using a 60x oil objective and 1 µm slice photographs through the z-axis. The ratio of double labeled/total BrdU+ cells examined was multiplied by the total BrdU+ cell population to obtain the estimated total number of BrdU+DCX+ cells per animal.