Microglia Isolation by Immunostaining Coupled Cell Sorting: An Immunostaining Method to Isolate Microglia from Zebrafish Brain Cells Using Fluorescent Activated Cell Sorting

Under pathological conditions, activated microglia – specialized brain macrophages – help eliminate pathogens and cell debris. These microglia express unique receptors that differentiate them from other macrophages.

To isolate microglia, begin with a  brain cell culture derived from transgenic zebrafish, expressing specific fluorescent molecules in different cell types.

The culture contains a mixture of neurons – expressing red fluorescence, macrophages – expressing green fluorescence, and non-fluorescent microglia.

Now, add a blocking reagent and incubate the culture with intermittent agitation. The blocking reagent binds to the non-specific sites on the cells and reduces the background signal.

Next, add primary antibodies and incubate. These antibodies bind to specific receptors expressed on the microglia surface.

Centrifuge the culture and remove the supernatant containing unbound antibodies. Resuspend the cell pellet in a fresh medium.

Subsequently, add fluorescent-dye conjugated secondary antibodies and incubate. These antibodies bind to the primary antibodies and impart a red color to the microglia-antibody complex.

Centrifuge the culture and remove unbound antibodies. Resuspend the pellet in a fresh medium.

Filter the cells to remove any debris.

Using a fluorescent activated cell sorter or FACS, sort the population of microglia cells based on their cell size and fluorescence.

The isolated microglia can be used for further applications.