Experimental methods to measure hormone levels in nuptial pads

Measuring hormone levels associated with nuptial pad development is crucial for research. Since the pads are a response to systemic hormones, scientists often measure hormone levels in the blood, but can also analyze the tissue itself.

Key experimental methods include:

1. Immunoassays (RIA and ELISA): These are the most common techniques. A blood sample is taken from the frog, and the plasma is analyzed using Radioimmunoassay (RIA) or Enzyme-Linked Immunosorbent Assay (ELISA). These methods use specific antibodies to detect and quantify the concentration of hormones like testosterone, DHT, and estradiol.
2. Tissue Homogenization and Extraction: A small tissue sample (biopsy) is taken from the nuptial pad. The tissue is ground up (homogenized), and the hormones within are extracted using solvents. The concentration in the extract is then measured using RIA or ELISA. This gives a direct measure of hormones present in the target tissue.
3. Liquid Chromatography-Mass Spectrometry (LC-MS): This is a highly advanced and sensitive method that can separate and accurately measure the levels of multiple different hormones and their metabolites from a single small sample of blood or tissue.
4. Immunohistochemistry: This technique uses fluorescently-labeled antibodies to visualize the location of hormone receptors (like androgen receptors) within thin slices of nuptial pad tissue viewed under a microscope. This shows where the hormones are acting, rather than how much is present.