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

Abstract

Source: Mazzolini, J. et al. Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains. J. Vis. Exp. (2018)

In this video, we isolate zebrafish microglial cells via immunostaining-coupled cell sorting. The isolated microglia can be used in downstream experiments to understand their role in brain pathologies.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Microglia Immunostaining

NOTE: All steps are performed at 4 °C.

1. Resuspend the cell pellet obtained from brain culture cells with 0.3 mL of Media A + 2% normal goat serum or NGS. Split them in 3 x 1.5 mL tubes: one for unstained cells to measure auto-fluorescence from cells of interest, second for the secondary antibody (1/200) to measure the non-specific binding of the secondary antibody to microglia, and third a....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Hepes	Gibco	15630-056
D-Glucose	Sigma	G8644-100ML
HBSS 1X	Gibco	14170-088
Normal goat serum (NGS)	Cell Signalling	5425S
35 μm cell strainer cap	BD	352235
FACS tubes	BD	352063
Low Endotoxin, Azide-Free (LEAF)	Biolegend	101321
Alexa Fluor 647 Goat Anti-Mouse IgG (H+L)	Life Technologies	A11008
Anti-4C4	Courtesy of Catherina Becker (University of Edinburgh)
FACS sorter FACSAria II	BD	QMRI, FACS facility