Name: immunofluorescence staining using iba1 and tmem119 for microglial density, morphology and peripheral myeloid cell infiltration analysis in mouse brain
Uploaded: 2019-10-27T15:00:00
Description: Watch this Scientific Journal Video about Immunofluorescence Staining Using IBA1 and TMEM119 for Microglial Density, Morphology and Peripheral Myeloid Cell Infiltration Analysis in Mouse Brain at JoVE

We are grateful to Nathalie Vernoux for her guidance and assistance with the experiments. We would also like to thank Drs. Emmanuel Planel and Serge Rivest for the use of their fluorescence and confocal microscopes, respectively. This work was partly funded by scholarships from Mexican Council of Science and Technology (CONACYT; to F.G.I), Fondation Famille-Choquette and Centre th&#233;matique de recherche en neurosciences (CTRN; to K.P.), Fonds de Recherche du Qu&#233;bec - Sant&#233; (to M.B.), and Shastri Indo-Canadian Institute (to K.B.), as well as a Discovery grant from Natural Sciences and Engineering Research Council of Canada (NSERC) to M.E.T. M.E.T. holds a Canada Research Chair (Tier II) of Neuroimmune Plasticity in Health and Therapy.

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 Universit&#233; Laval.
1. Immunostaining
<ol>
	<li>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 &#181;L of phosphate-buffered s...

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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...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/60510/immunofluorescence-staining-using-iba1-tmem119-for-microglial-density">Immunofluorescence Staining Using IBA1 and TMEM119 for Microglial Density, Morphology and Peripheral Myeloid Cell Infiltration Analysis in Mouse Brain</a>. An&#160;author&#160;name was&#160;updated.
The&#160;name&#160;was&#160;corrected from:
Maude&#160;Bordelau
to:
Maude&#160;Bordeleau

An erratum was issued for:&#160;<a href="https://www.jove.com/t/60510/immunofluorescence-staining-using-iba1-tmem119-for-microglial-density">Immunofluorescence Staining Using IBA1 and TMEM119 for Microglial Density, Morphology and Peripheral Myeloid Cell Infiltration Analysis in Mouse Brain</a>. An&#160;author&#160;name was&#160;updated.

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

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 physiological conditions3. Microglia are equipped with an arsenal of receptors that allow them to perform immunological tasks and respond to changes in the brain milieu, to cell death, or to tissue damage. In addition, they exert key physiological functions, notably in synaptic formation, maintenance, and elimination4,5.
Among the available markers used to study microglia, ionized calcium binding adaptor molecule 1 (IBA1) is one of the most widely used. IBA1 is a calcium binding protein that provides exceptional visualization of microglial morphology, including fine distal processes, as confirmed by electron microscopy6. This tool has been instrumental in characterizing microglial transformation, formerly called &#34;activation&#34;, in a vast array of animal disease models7,8,9. In the presence of neuroinflammation, the microglial response includes: microgliosis that is defined as an increase in cellular density, changes in distribution that sometimes result in clustering, enlargement of the cell body, as well as thickening and shortening of processes associated with more ameboid shapes10,11,12,13.
Immunostaining is limited by the availability of antibodies directed against specific markers. Importantly, IBA1 is expressed by microglia but also by peripheral macrophages that infiltrate the brain14. While observation of IBA1-positive cells inside the brain has become a marker of microglia in this research field, peripheral macrophage infiltration has been reported under various conditions, even marginally in the healthy brain15,16,17,18. Consequently, the use of IBA1 alone does not allow selective visualization of microglia. In addition, macrophages adopt molecular and morphological features of resident microglia once they have infiltrated the brain, thus hindering differentiation19. This represents a challenge when investigating the function of both microglia and infiltrating macrophages.
While microglia and peripheral macrophages have distinct origins (e.g., from the embryonic yolk sac and bone marrow, respectively20,21), there is an increasing number of findings indicating that the two cell populations exert different roles in the brain19. It is thus crucial to use methods that discriminate between these two populations without invasive manipulations (i.e., bone marrow chimeras or parabiosis) that can modulate their density, distribution, morphology, and function. TMEM119 has emerged as a microglia-specific marker across health and disease conditions22. When combined with IBA1, this marker becomes useful for differentiating these cells from infiltrating macrophages, which are TMEM119-negative and IBA1-positive. While it is developmentally regulated, TMEM119 is expressed as early as postnatal days 3 (P3) and 6 (P6), steadily increasing until reaching adult levels between P10 and P1422. IBA1 is expressed as early as embryonic day 10.5 (E10.5)23. The proposed double labeling protocol is thus useful to study these two populations throughout postnatal life.
This protocol provides a step-by-step immunostaining procedure that allows discrimination between microglia and peripheral macrophages. It also explains how to conduct a quantitative analysis of microglial density, distribution, and morphology, as well as analysis of peripheral macrophage infiltration. While the investigation of microglia and peripheral macrophages is useful on its own, this protocol further allows localization of neuroinflammatory foyers; thus, it also serves as a platform to identify specific regions to investigate, with the use of complementary (yet, more time- and resource-consuming) techniques.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 488 donkey anti-mouse</td>
			<td>Invitrogen/Thermofisher</td>
			<td>A21202</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 goat anti-rabbit</td>
			<td>Invitrogen/Thermofisher</td>
			<td>A11011</td>
			<td></td>
		</tr>
		<tr>
			<td>Biolite 24 Well multidish</td>
			<td>Thermo Fisher</td>
			<td>930186</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>EMD Millipore Corporation</td>
			<td>2930</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Sigma-Aldrich</td>
			<td>C0759-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI Nuceleic acid stain</td>
			<td>Invitrogen/Thermofisher</td>
			<td>MP 01306</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Brush</td>
			<td>Art store</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount-G</td>
			<td>Southern Biotech</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin from coldwater fish skin</td>
			<td>Sigma-Aldrich</td>
			<td>G7765</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope coverglass</td>
			<td>Fisher Scientific</td>
			<td>1254418</td>
			<td></td>
		</tr>
		<tr>
			<td>Microslides positively charged</td>
			<td>VWR</td>
			<td>48311-703</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal mouse Anti-IBA1</td>
			<td>Millipore</td>
			<td>MABN92</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2H2PO4&#183;H2O</td>
			<td>BioShop Canada Inc.</td>
			<td>SPM306, SPM400</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>BioShop Canada Inc.</td>
			<td>SPD307, SPD600</td>
			<td></td>
		</tr>
		<tr>
			<td>NaBH4</td>
			<td>Sigma-Aldrich</td>
			<td>480886</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>S642500</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal donkey serum (NDS)</td>
			<td>Jackson ImmunoResearch laboratories Inc.</td>
			<td>017-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum (NGS)</td>
			<td>Jackson ImmunoResearch laboratories Inc.</td>
			<td>005-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm-M</td>
			<td>Parafilm</td>
			<td>PM-999</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit monoclonal Anti-TMEM119</td>
			<td>Abcam</td>
			<td>ab209064</td>
			<td></td>
		</tr>
		<tr>
			<td>Reciprocal Shaking bath model 25</td>
			<td>Precision Scientific</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tris buffer hydrochloride</td>
			<td>BioShop Canada Inc.</td>
			<td>TRS002/TRS004</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P7949-100ML</td>
			<td></td>
		</tr>
	</tbody>
</table>

immunofluorescence staining using iba1 and tmem119 for microglial density, morphology and peripheral myeloid cell infiltration analysis in mouse brain

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.

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&#8722;C). This staining allows determination of microglial density and distribution and identification of microglial clusters (Figure 1<stron...

Watch this Scientific Journal Video about Immunofluorescence Staining Using IBA1 and TMEM119 for Microglial Density, Morphology and Peripheral Myeloid Cell Infiltration Analysis in Mouse Brain at JoVE

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

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.

This is a protocol for the dual visualization of microglia and infiltrating macrophages in mouse brain tissue. TMEM119 (which labels microglia ...

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