I am working on estradiol neuroprotection impact and measuring its concentration in hippocampal female rats in their diestrus phase by 17beta-Estradiol ELISA (Diagnostics Biochem Canada). To extract estradiol from hippocampus the tissue was homogenized in 150µl PBS and centrifuged at 15,000g for 30 min at 4ºC and supernatants were collected. The problem is almost no level of estradiol was detected through this method.
I would think that you will never be able to measure endogenous estradiol concentration from brain extracts using ELISA.
Either you do the other way around: test the effect of ading exogenous estradiol in a model, or you move to another approach for which you will need help from experts working with HPLC-Mas spect. If you make this move you also need to change totally the way you process the tissue.
HPLC-Mas spec are the only reliable methods to detect estradiol in samples you are intending to investigate.
You may alternatively try to look for an enzyme based staining (which may generate some color). Then color intensity or densitometry can be done to give you a rough estimate.
But I agree with Prof. Franco every single bit.
All the best for your experiments !
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