Hi All,
Recently I want to study neurogenesis, and try to use Edu as a cell marker for proliferation. However the edu not working very well so I decided change it to other markers, such as Ki67 or PCNA. Some friends told me ki67 only label S phase, as neuron no longer divided, I won't get Ki67/Tuj1/NeuN labeled cells in my CNS neurons culture. But wiki said Ki67 will label all cell phase except G0, which confused me a lot. Any one get Ki67 labeled neurons maker (i.e Tuj1/NeuN) yet?
I have not worked with neurons but you are correct, Ki67 marks every stage of the cell cycle except G0. As such you should get labelling if your cells are proliferating. However EdU marks cells that are entering S phase during your chase period, as it gets incorporated into DNA as the cells divide. Hope that helps!
Ki67 should not co-label with NeuN, as they are by definition post-mitotic. Also bear in mind that as Ki67 is expressed thorough the cell cycle, it will label more cells than just EdU. What protocol are you using for the EdU? It's normally pretty reliable.
I have experience both on Ki67 and Edu, as Wiki says, Ki67 stains all proliferating cells except resting G0, and of course Edu stains S phase only, but combining PI or Hoechst, Edu can split cells into G1, S and G2M phases which is much more informative. I use Click-iT® EdU Alexa Fluor® 488 Imaging Kit from Invitrogen, the most funny is the protocol from the company didn't well, I made my homemade buffers (very simple, just VitC, tris and CuSO4) according to the reference paper (http://www.ncbi.nlm.nih.gov/pubmed/18272492), it worked beautifully and very stable, Edu and Azide-AF488 can be titrated 20 fold and still worked well.
Hope this may help.
Yes, the kit from Invitrogen uses far more of the azide than is required. I've used both home-made solutions and the kit, both work for me.
Hi, I have a related question - I used the ClickIT EdU A488 kit on neural stem cells in culture. After fixing the cells, I detected EdU with the kit and then immunostained the culture with Ki67 and Pax6. I see that all Ki67+ cells are EdU+ and I was not expecting that since EdU marks only S-phase cells. There are no cells that are Ki67+/EdU-. I had treated the cells with 10uM EdU for 4h followed by a 24h and 48h chase. Is the EdU detection hampering downstream immunoctyochemistry?
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