Mouse rat estradiol elisa

Circulating white blood cell counts after 6 weeks of E₂ supplementation (vs. age-matched controls) were determined using a Hemavet 950DS (Drew Scientific, Miami Lakes, FL). E₂ levels in serum collected 2 or 6 weeks post pellet placement, and stored at −80°C prior to assay, were assayed by the University of Virginia Center for Research in Reproduction Ligand Assay and Analysis Core using a commercially available mouse/rat estradiol ELISA (Calbiotech, El Cajon, CA)[38 (link)]. Markers of bone formation (rat/mouse P1NP) or resorption (mouse TRAcP 5b) were assayed in fasting serum collected 2 weeks after start of E₂ supplementation (vs. age-matched controls) using commercially available ELISAs (Immunodiagnostic Systems, United Kingdom) [22 (link),25 (link)].

Vaginal cytology (see Supplemental Figure 1 for representative images) took place at least 4 days after the last behavior test, between 11:00 AM and 1:00 PM every weekday for 2 weeks. Mice were group housed for the duration of the vaginal cytology measurements. Q-Tips soaked in PBS were used to gently swab the vaginal opening and rolled out onto a glass slide. Slides were set out to dry, and then 2 drops of crystal violet stain were applied. Slides were immediately coverslipped and put into a drawer for at least 2 minutes to protect the stain from light. Slides were examined under a microscope within minutes of staining, and estrus cycle phase was determined by analyzing the dominant composition of cells within the swab(18 (link)). Proestrus was identified by a predominance of nucleated, with some cornified epithelial cells. Estrus was identified by a predominance of cornified epithelial cells. Metestrus was determined by cornified epithelial cells as well as polymorphonuclear leukocytes. Lastly, diestrus was determined by primarily polymorphonuclear leukocytes. Mice were determined to be peri-estropausal/approaching reproductive senescence with irregular cycles (data not shown) and plasma estradiol levels were below the limit of detection (measured via Calbiotech Mouse/Rat Estradiol ELISA, catalog # ES180S-100ELISA, 3pg/ml sensitivity; data not shown).