Evos fl auto 2.0 microscope

To examine estrous cycle state vaginal lavages were performed throughout FLX/vehicle treatment, after completing each behavioral test, and prior euthanasia. In order to collect the samples, a pipet was filled with ddH₂O, placed at the opening of the mouse’s vaginal canal (without penetration) with ddH₂O gently expelled and suctioned back (Byers et al. 2012 (link); McLean et al. 2012 (link)). Samples were then placed on a slide warmer to dry for approximately 5 minutes and imaged under a EVOS FL Auto 2.0 microscope (Thermofisher Scientific) at 10× magnification. Estrous phase was identified by the presence or absence of nucleated epithelial cells, cornified epithelial cells, and leukocytes (Byers et al. 2012 (link); Felicio et al. 1984 (link)). Mice in proestrus displayed mostly nucleated and some cornified cells (Figure 1B). Estrus was recorded as the presence of mostly cornified epithelial cells, with the presence of a few nucleated cells in early estrus (Figure 1B). Metestrus was determined by the presence of cornified epithelial cells and polymorphonuclear leukocytes (Byers et al. 2012 (link)), while mice in diestrus contained mainly polymorphonuclear leukocytes with few epithelial cells being present (Figure 1B).

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).

Vaginal lavages were performed daily during the stress paradigms and two weeks prior to behavioral testing to ensure mice were cycling throughout all four stages of the estrous cycle regularly. After completing each behavioral test, vaginal smears were collected to assess the estrous state mice were in during the behavior test. Samples were collected via a pipette filled with ddH₂O gently expelled and placed at the vaginal canal opening (without penetration). Samples were suctioned back into the pipette, placed on a microscope slide, and dried on a slide warmer before imaged with an EVOS FL Auto 2.0 microscope (Thermofisher Scientific) at 10x magnification (40, 41). Estrous phases were identified by the presence or absence of nucleated epithelial cells, cornified epithelial cells, and leukocytes (41, 42 Figure 3B).