To quantify neurogenesis in the dentate gyrus, instead of doublecortin IHC, can I use doubecortin ELISA by using hippocampal tissue homogenate?. I can not find any reference articles for detection of neurogenesis by ELISA . I am facing difficulty in trouble-shooting DCX IHC.
Hi, Rajesh.
Most of cell differentiation biomarkers are cytoskeletal proteins present abundantly. Their structures are not easy to access once they are fixed or even in live tissue due to tight and complex structures. Therefore, you cannot properly extract DCX from the associated MT and many other proteins to do fluid-based ELISA assay.
Check your IHC procedure again. Tissue fixation, washing the fixative, thickness, primary DCX antibody, 2nd Ab are matching and freshly-made or whether they are stored properly until your use, etc. I suggest cryostat of fresh tissue, although this procedure needs similar precautions. If you have verified all parameters above, still not working. Then I suggest low power of microwave oven to loosen or demask some of the molecules within the tissue.
If the aim is quantitation of DCX expressed, then do Western blot of the same region of DG among the groups. Quick freeze the same amount of the DG region in LN2, Grind the tissue with a mortar and pestle completely. Transfer into small eppen-tube, weight, add lysis buffer according to the weights in the same ratio of wt/vol to adjust tissue amount again.Then, follow the SDS-sample buffer for PAGE gel running. This is a semiquantitation.
That will be a good scientific data, in case of impossibility to measure every molecules of DCX expressed.
You could do immunoblot of SDS-sample buffer without BPB dye.
Good luck to you!
See also the link
https://www.researchgate.net/post/Neurogenesis_in_chickens_dentate_gyrus-can_anyone_help
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