Vaginal Lavage Estrous Cycle Staging

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).