Cy 5 goat anti rabbit

The effects of FLX treatment on cell proliferation were assessed across a total of 12 sections (every sixth section) of the hippocampus. Slides were washed in 1% Triton X-100 PBS for 5 minutes before undergoing three PBS washes. Next, slides were incubated in warm citrate buffer for 30 minutes and then washed three times in PBS. Slides were then transferred to an opaque moisture chamber (TedPella) for the blocking and overnight incubation step. Slides were blocked for 1 hour in 10% normal goat serum (NGS) diluted in PBS before being incubated overnight at 4°C in anti-rabbit Ki67 (1:250; polyclonal abCam, ab15580) diluted in 2% NGS PBS. Following 18 hours of incubation slides were washed 3 times in PBS before being incubated at room temperature for 2 hours in CY-5 goat anti-rabbit (1:1000, Invitrogen, Thermo Fisher Scientific, A10523) diluted in 2% NGS PBS. Next slides were washed with PBS then counterstained with DAPI (1:15000) for 15 minutes. Finally, slides were washed with PBS and cover slipped using the mounting medium Prolong Diamond (Thermofisher Scientific). Fluorescent images were taken using a EVOS FL Auto 2.0 microscope (Thermofisher Scientific) at 10× or 40× magnification, where Ki67⁺ cells overlayed with DAPI (Thermofisher Scientific) across the 12 sections of hippocampus were collected and counted via an observer blind to treatment (Supplemental Figure 2A–E).

12 hippocampal sections (every sixth section) for doublecortin (DCX) were stained using the primary antibody doublecortin anti-goat (1:500; Life technologies; 481200) and secondary antibody CY-5 goat anti-rabbit (1:1000, Invitrogen, Thermo Fisher Scientific, A10523). Fluorescent images were taken using using a EVOS FL Auto 2.0 microscope (Thermofisher Scientific) at 10× or 40× magnification, where DCX⁺ cells across the 12 sections of hippocampus were collected and counted by an observer blind to treatment (Supplemental Figure 2F–O). Following imaging, DCX⁺ cells were counted and subcategorized according to their dendritic morphology: DCX⁺ cells with no tertiary dendritic processes and DCX⁺ cells with complex, tertiary dendrites. The maturation index was defined as the ratio of DCX⁺ cells possessing tertiary dendrites over the total DCX⁺ cells.