I think you will find a good protocol in this article:

Erythropoietin dampens injury-induced microglial motility

Article in Journal of cerebral blood flow and metabolism: 35(8) · May 2015

Dear Tabasum Sidiq ,

There are several methods to isolate microglia from mouse brain in the literature. Most of them are based on the shaking method in which microglia is isolated by shaking the culture flasks of mix glia culture with a speed about 250-300 rpm for 1-2 hours . However, I recommend you to isolate microglia by immunopanning method by using Anti-CD45 antibody, due to your specific experimental purpose. By using this method, you can do your RNA-Seq immediately after the acute isolation of microglia, which should be more  similar with in vivo condition than the shaking method. For detailed protocol, please see Ben Barres's paper: An RNA-Sequencing Transcriptome and Splicing Database of Glia, Neurons, and Vascular Cells of the Cerebral Cortex, JNS, 2014.

I have successfully isolated microglia from adult rat brains using: Frank et al. 2006 (Journal of Neuroscience Methods 151: 121–130).    It is much faster than other methods that I have used.

Thank you all for sharing the protocols.

Dear Tabasum,

here's a video tutorial for the isolation of microglia from adult mouse brain.

https://youtu.be/XYg7KSsZZys

For mice younger than P7 you can use a similar protocol leaving out the myelin removal step.

Good luck!

Sandra