Hello! I think I can help you as I needed to stablish a microglia isolation protocol in my lab some months ago. From what I read in the literature there are three main ways to do it: by column separation with beads (like with Iba1 antibodies), column separation by density or the way I did it: by culturing a mixed glia culture and then separate by tapping or shaking.

In brief I proceed with this protocol:

- I use P0-P1 pups (but i guess even with E18-20 pups should be the same, you just proceed with the C section and take the embryos).

- I decapitate the pup/embryo and spray ethanol on the head.

- under the binocular microscope I cut the skin and open the skull. I remove the brain and place it in a petri dish with HBSS or cell culture medium.

- I take away the meninges very carefully with tweezers (very important step to avoid contamination of circulating macrophages).

- Here in my case I isolate the hippocampus as I am interested in hippocampal microglia but If you want more quantity I would go for the cortex as well.

- I place all the dissected hippocampus in a falcon tube and I mechanically dissociate the tissue by pipetting up and down around 30 times (some protocols use some enzymes to dissociate but I do not do it and have nice results).

- I filter the solution with a 33um filter to eliminate debris and big chunks of tissue.

- I add as medium as necessary and I plate the cells in poly-D-lysine coated P75 flasks with a density of 2 pups per flask. (4 hippocampi per flask). I use the medium in which only glia can grow: DMEM, 10% Horse Serum, 1% Pen/strep. The low density is very important as will allow microglia to really form a layer on top of astrocytes.

- One day after plating I change all the medium. You should observe cells adhered to the surface that should only be microglia and astrocytes who survived.

- For 10 to 15 days I let the cultures grow changing the medium every 2-3 days but only 50% of the medium (this step is essential as microglia need factors released by astrocytes to proliferate).

- When confluency is reached and I also observe a layer of microglia on top of astrocytes I proceed with microglia separation: I shake the flasks for 2h 100rpm (ensure 37ºC and 5% CO2 conditions or microglia will die).

- I take away the supernatant, centrifuge and plate in coated 24-well plates at a density of 100 000 cells/well. After 24h you see microglia only in the wells with nice morphology. VERY IMPORTANT HERE: after centrifugation keep the medium of the mixed glia culture and use it to culture microglia as they need conditioned medium from astrocytes to survive.

I hope this helps :)