Immunofluorescence Staining Using IBA1 and TMEM119 for Microglial Density, Morphology and Peripheral Myeloid Cell Infiltration Analysis in Mouse Brain

Summary

This protocol describes a step-by-step workflow for immunofluorescent costaining of IBA1 and TMEM119, in addition to analysis of microglial density, distribution, and morphology, as well as peripheral myeloid cell infiltration in mouse brain tissue.

Abstract

This is a protocol for the dual visualization of microglia and infiltrating macrophages in mouse brain tissue. TMEM119 (which labels microglia selectively), when combined with IBA1 (which provides an exceptional visualization of their morphology), allows investigation of changes in density, distribution, and morphology. Quantifying these parameters is important in providing insights into the roles exerted by microglia, the resident macrophages of the brain. Under normal physiological conditions, microglia are regularly distributed in a mosaic-like pattern and present a small soma with ramified processes. Nevertheless, as a response to environmental factors (i.e., trauma, infection, disease, or injury), microglial density, distribution, and morphology are altered in various manners, depending on the insult. Additionally, the described double-staining method allows visualization of infiltrating macrophages in the brain based on their expression of IBA1 and without colocalization with TMEM119. This approach thus allows discrimination between microglia and infiltrating macrophages, which is required to provide functional insights into their distinct involvement in brain homeostasis across various contexts of health and disease. This protocol integrates the latest findings in neuroimmunology that pertain to the identification of selective markers. It also serves as a useful tool for both experienced neuroimmunologists and researchers seeking to integrate neuroimmunology into projects.

Introduction

Whether acute or chronic, neuroinflammation is tightly influenced by microglia, the resident macrophages of the brain. Visualizing microglia through immunostaining is valuable for the study of neuroinflammation with the use of light microscopy, a highly accessible technique. In homeostatic conditions, microglia are typically distributed in a nonoverlapping, mosaic-like pattern. They exhibit small somas that extend ramified processes¹, which sometimes contact one another². Microglial ramified processes dynamically survey the brain parenchyma, interacting with neurons, other glial cells, and blood vessels during normal phy

Protocol

All experimental procedures were performed in agreement with the guidelines of the Institutional Animal Ethics committees, in conformity with the Canadian Council on Animal Care and the Animal Care Committee of Université Laval.

1. Immunostaining

1. Select three mouse brain sections containing the region of interest (ROI) (i.e., the hippocampus) with the help of a brain atlas. Place the sections in a plastic multi-well plate and cover them with 350 µL of phosphate-buffered saline (PBS) (Table 1).
   NOTE: For optimal results, the brains should be perfused with 4% paraformaldehyde and cut to a thicknes

Results

Figure 1 shows the co-labeling of microglia using IBA1 and TMEM119 in a coronal section of the dorsal hippocampus imaged at 20x by fluorescence microscopy. A successful staining reveals microglial cell bodies and their fine processes (Figure 1A−C). This staining allows determination of microglial density and distribution and identification of microglial clusters (Figure 1

Discussion

This protocol can be divided in two critical parts: quality of the staining and analysis. If the staining is not optimal, it will fail to represent microglial cells adequately, thus affecting the density, distribution, and morphology measurements. In addition, the proportion of infiltration peripheral myeloid cells may be underestimated. This is an optimized version of the staining protocol, but there are several factors that may result in suboptimal images. Even though the perfusion of the animal is not included in this...

Materials

Name	Company	Catalog Number	Comments
Alexa Fluor 488 donkey anti-mouse	Invitrogen/Thermofisher	A21202
Alexa Fluor 568 goat anti-rabbit	Invitrogen/Thermofisher	A11011
Biolite 24 Well multidish	Thermo Fisher	930186
Bovine serum albumin	EMD Millipore Corporation	2930
Citric acid	Sigma-Aldrich	C0759-500G
DAPI Nuceleic acid stain	Invitrogen/Thermofisher	MP 01306
Fine Brush	Art store
Fluoromount-G	Southern Biotech	0100-01
Gelatin from coldwater fish skin	Sigma-Aldrich	G7765
Microscope coverglass	Fisher Scientific	1254418
Microslides positively charged	VWR	48311-703
Monoclonal mouse Anti-IBA1	Millipore	MABN92
Na2H2PO4·H2O	BioShop Canada Inc.	SPM306, SPM400
Na2HPO4	BioShop Canada Inc.	SPD307, SPD600
NaBH4	Sigma-Aldrich	480886
NaCl	Fisher Scientific	S642500
Normal donkey serum (NDS)	Jackson ImmunoResearch laboratories Inc.	017-000-121
Normal goat serum (NGS)	Jackson ImmunoResearch laboratories Inc.	005-000-121
Parafilm-M	Parafilm	PM-999
Rabbit monoclonal Anti-TMEM119	Abcam	ab209064
Reciprocal Shaking bath model 25	Precision Scientific	-
Transfer pipette
Tris buffer hydrochloride	BioShop Canada Inc.	TRS002/TRS004
Triton-X-100	Sigma-Aldrich	T8787
Tween 20	Sigma-Aldrich	P7949-100ML