Aldh1L1 and/or GFAP. Also see:

http://www.jneurosci.org/content/28/1/264.full

Hi, you can use Phospho-Ser10 Histone 3 antibody to detect cell proliferation. The phosphorylation is specific for mitotic cells. Other possibilities are Cyclin A or Cyclin B which are also specific for cycling cells.

Hi Matteo,

I work on adult neural stem cell research and hope I can help you a bit. We measure proliferation in vitro by immnuofluorescence. First of all, I am not sure if your primary cultures have only astrocytes, or astrocytes together with other cell lineages (e.g. neurons and oligodendrocytes). If your case is only astrocytes, you can use cell proliferation markers, such as Ki67 or phosho-ser 10 histone 3 antibody as Christian was suggesting. This last antibody will label specifically cells in mitotic phase, so you will find less cells positive for this marker than for Ki67. For primary cultures with a mixture of cell types, I would suggest to do double immunofluorescence for GFAP (or S100beta, Aldh1L1, etc) together with Ki67 to be able to count the percentage of astrocytes that are proliferating. BrdU is also a good method, you will need to treat your cells with HCl treatment after fixation and before antibody incubation to get the staining work.

For western blot, the expression of cyclins do not correlate 100% to the rate of cell proliferation, With some particular cyclins it can be inversely correlated. I would be careful if you want to do western blot to detect proliferation in your astrocytes. I suggest you to combine western blot data and immunofluorescence to be sure about your results.

Hope my answer helped your research,

Ale

Thanks guys,

Alejandro, I do work with a mixed primary culture so I have both neurons and all type of glial cells plated together. We have GFAP and BrdU in the lab so I will go most likely for that. I have experience with immunostaining but I have never used HCl, what's the reason of that?

Thanks,

Matteo

Matteo, you need to partly digest the DNA in the nucleus in order to expose the incorporated BrdU so that it can be reached by an antibody. Unfortunately, this HCl-treatment precludes staining with many other antibodies. I have no clue whether this applies to your GFAP ab. If it does not work you can buy a EdU kit. EdU is a analog of BrdU but it can be visualized by adding a fluorophore directly to the incorporated nucleotide using Click-chemistry. So there is no need for HCl and ab staining can be done as usual. Unfortunately, the kit is a bit pricy...