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

To immunostain microglia, use 0.3 milliliters of Media A plus 2% NGS to resuspend the cell pellet. Then, split the cells between three 1.5-milliliter tubes: one for unstained cells to measure autofluorescence, one to measure the nonspecific binding of a secondary antibody to microglia, and a third as a test.

To all the tubes, add 1% Low Endotoxin, Azide-Free (LEAF) to block CD16/CD32 interactions with the Fc domain of immunoglobulins. Then, incubate the cells for 10 minutes with gentle agitation every 5 minutes.

Next, to the third tube, add the 4C4 antibody and incubate it for 30 minutes with gentle agitation every 10 minutes. Then, spin the tubes at 300 x g and 4 degrees Celsius for 10 minutes.

After discarding the supernatant, wash the pellet once with 0.5 milliliters of Media A plus 2% NGS before spinning the tubes again. Resuspend the cell pellet with 0.5 milliliters of Media A plus 2% NGS, then add 1% LEAF and incubate the cells for 10 minutes with gentle agitation every 5 minutes.

To tubes 2 and 3, add secondary antibodies and place the tubes in the dark. After spinning the tubes and discarding the supernatant, use 0.5 milliliters of Media A plus 2% NGS to wash the samples twice, then resuspend the cell pellets with 1 milliliter of Media A plus 2% NGS.

Run the cell suspension through a 35-micron cell strainer cap and transfer it into cold 5-millimeter FACS tubes on ice protected from light. Finally, carry out FACS sorting and RNA extraction according to the text protocol.