Since both markers can be used to check the proliferation of neural stem cells, I was wondering what is the difference in using them? Is there a difference in the method using it or price? Can someone please help me out with which one I should pick as a good proliferative marker for the hippocampal cell line?
BrdU is used as a general proliferation marker showing DNA synthesis. Nestin is an intermediate filament mainly expressed in brain stem cells and precursor cells. I would suggest to use BrdU (or EdU, Click it cell Proliferation Kit) to show cell proliferation, because nestin is also expressed in e.g. mature astrocytes to little extent and these cells do not proliferate. If you are sure to have brain precursor or stem cells, it would be enough to confirm the proliferative identity of cells by BrdU/EdU. But since you talk about a cell line, I would simultaneously test for nestin expression to see what cell type you actually have.
ahh ok..thanks so much Susanne. ill go through more about the techniques using them now..if you could provide me some pdf files of those techs you are following, i would really appreciate that.
Nestin is an endogenous marker and BrdU has to be injected. With BrdU you can mark new born cells at a certain timepoint of interrest, with Nestin you are not sure about the age of the cell.
As far as I know, to stain braintissue with BrdU antibodies, you need quite severe treatment (heating and acid treatment) which makes double staining difficult. In general for rodent studies 2 stainings are used, one with BrdU and one with an endogenous marker, which is often Ki-67.
I agree with James and Dirk-Jan, Ki-67 is also a reliable marker for proliferative cells. We are using theClick-iT® EdU Alexa Fluor® 488 Imaging Kit (http://www.lifetechnologies.com/order/catalog/product/C10337). With this you confirm DNA synthesis and are able to do a co-staining of endogenous marker.
Phosphorylated Vimentin (4A4) is a M-phase specific marker that allows you to count the nucleus of mitotic radial glia. This is useful to count the rate of neural progenitor cell proliferation. I have used Ki67 and Nestin which are expressed through S phase and M phase of the cell cycle making your work a bit more difficult. Another nice thing about counting M phase is that all the radial glia in the ventricular zone will line up on the ventricular surface during mitosis.
there are also PCNA and phospho-Histone3 http://www.nature.com/onc/journal/v20/n24/full/1204326a.html
Note that BrdU may also be incorporated into chromatin without cell division, and this may be a sign of cells under stress undergoing DNA repair. Nestin-positivity is not a sign of proliferation, though it may be indirect evidence that cells are still capable to proliferate. The comment about soe initial difficulty working out IHC for detecting BrdU is absolutely right, what further complicates the IHC is method of embededing, if you used paraffinn, you need to unmask, and do it right. If you use lightly PFA-fixed frozens, under- and over treatment is equally bad, one of my students spent quite some time working out conditions. Acid has to be right concentration, and i believe freshly diluted. Not all anti-BrdU antibodies work well on paraffin vs frozens. I suggest ask for help and join somebody who is doing such detection regularly, and do it side by side, same antibody, same acid, track acid treatment with timer (seconds matter)
- Badges
- Science method