Hi Kevin.

Please share the protocol you followed for isolating primary astrocytes from mice. How did you standardize your primary astrocyte culture protocol? I have worked on isolating and conducted IF analysis of GFAP and have found the same problem in the beginning. I suppose there may be microglial and oligodendrocytes contamination.

I followed this protocol- this worked quite well for me. Article Isolation and Culture of Mouse Cortical Astrocytes

Thanks

Hope it works.

Hi Kevin, How many days post dissection did you analyse your culture? In Astrocyte primary cultures you may still see a few microglial cells on the first days post dissection which should reduce over time. Nevertheless, you might still see some microglia even after a few weeks (despite of shaking them off and splitting the cells). But NeuN, should definitely be negative all the time, as neurons can not survive in the Astrocyte media and culture conditions (e.g. proper coating and supplements for neurons).

Samir Ranjan Panda. Thanks for your answer. The standardization I use is very similar to the one you use. The problem is that it is not a merge in some cells, which would indicate contamination, but it is almost total. I will attach the photo so that it can be better understood.

Behnam Mohammadi.I analyzed my culture three weeks after dissection. I don't think it's contamination. I will attach the photo for you to review.