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 ...

This protocol will show you how to stain microglia, perform quantitative density and morphology analysis as well as to quantify macrophage infiltration into the brain. This technique allows the researcher to measure changes in microglia distribution and morphology in a quantitative way and to differentiate microglia from infiltrating macrophages This analysis protocol can be easily applied to other animal models to measure changes in microglial distribution and morphology where anti-IBA1 and anti-TMEM119 antibodies are available. It is highly recommended to conduct the cell counts and traces blinded to the experimental condition and to be consistent in the way this procedure is performed.To begin this procedure, open an image processing program such Fiji or ImageJ that has the nearest neighbor distance plug-in installed. Open the 20X image if the scale is contained in the metadata of the file and not automatically set from the metadata. On the menu bar, select Analyze, Set Scale, then enter the correct information.To set the scale manually based on a scale imprinted on the image, select the Straight Line tool. Place the cursor on the edge of the scale and while pressing the Shift key, draw a line as close as possible to the scale on the image. Select Analyze, Set Scale.A window will pop up and put the correct known distance and unit of length to determine the pixel's per length unit and click OK.Next, select Image, Color, Make Composite to create a composite image of all channels. On the menu bar, select Analyze, Set Measurements. Check Area, Centroid and Perimeter.In the Redirect To dropdown menu, select the Open file then click OK.Go to Image, Color, Channel Tool to open the Channel tool. Use the Freehand Selection tool to draw a rough perimeter around the region of interest. Double click the Oval tool on the tool bar to enable the selection Brush tool.Make sure that the Enable Selection Brush box is checked and click OK.Using the selection brush, adjust the perimeter to best fit the region of interest, then press T on the keyboard throughout this area to arrow Y manager. Select Analyze, Measure or Press the M key and the Results window will pop up. Copy and paste the results into a data sheet and save the information regarding the area.After copying the area of the region of interest, you base the information from the Results window by clicking on it and pressing the Backspace key. Select the ROI Manager window, select the ROI Trace and change the name to match the image's name by pressing Rename. Click OK then right click the new name of the ROI Trace and save.Double click the Brush tool in the tool bar. Select the color black and the Brush size of 10. Make sure that the option Paint on overlay is unchecked.In the TMEM119 channel, carefully place a black dot on the center of the soma for each TMEM119 positive microglia. Place a white dot on the center of the cells that are not positive for TMEM119. Repeat the same procedure for all cells contained in the region of interest.Next, select Image, Color, Split Channel, a window for each channel will appear. Identify the channel that has the dot annotations and close the other two windows. To redirect the new split channel image, go to Analyze, Set Measurements.In the Redirect To drop down menu, select the Split Channel Image and click OK.Then select Image, Type, eight-bit. Go to Image Adjust and select Threshold. Adjust the sliders all the way to the left in both bars.This will leave only the black dots which will now appear white. Select the region of interest in the ROI Manager window. In the Menu bar, select Analyze, Analyze Particle.In the text box for Size, input one through 20. The box for Pixel units should remain unchecked while the box that's for Display results, Summarize and Add to Manager should be checked. Click OK to display the Summary window which will give the number of points.Copy and paste this information to the data sheet. Then, select plug-ins NND to show the NND window. Each number represents the distance each microglia has to the nearest neighboring microglia.Copy and paste all of this information to the data sheet. Go back to the Threshold window and adjust the top slider all the way to the right, which will leave all of the white dots visible, now appearing white. Select Analyze, Analyze Particle to display the Analyze Particles pop up window.Click OK to show the Summary window that provides the number of points. Copy and paste this information into the data sheet. Go to the ROI Manager and select all the points.Then right click and Save with the image's name. This will allow saving of all the points in a ZIP file. First, open the 4DX image files.A pop up window will appear asking if the images should be opened in a stack. Click OK.Next, select Image, Stacks, ZProject to open the Z Projection window. Include all slices from the first through last slice.Ensure that the Max Intensity is selected under Projection Type and click OK.Click on the new window that contains the ZProject. Select Image, Colors, Split Channels and conduct the traces on the images of the IBA1 channel. In the Menu bar, select Analyze, Set Measurements.Check the Area, Centroid and Perimeter. In the Redirect To drop down menu, select the open file and click OK.To set the scale manually based on a scale imprinted on the image, select the Straight Line tool. Place the cursor on the edge of the scale and while pressing the Shift key, draw a line as close as possible to the scale on the image.Select Analyze, Set Scale. A window will pop up. Input the correct known distance and unit of length to determine the Pixels per Length unit and click OK.To measure the soma size in the IBA1 channel, draw a rough perimeter of the soma with the Freehand Selection tool.Double click the Oval tool on the toolbar to enable the Selection Brush tool. Make sure that the Enable Selection Brush box is checked and click OK.Using the Selection Brush, adjust the trace to best fit the soma. Zooming in will enable precision during this step.Next, press the T key to add the soma trace to the ROI Manager. Select Analyze, Measure or press the M key to display the results. Copy and paste these results to a data sheet.Go to the ROI Manager window, select the region of interest and change the name to match the image's name by pressing Rename. Right click on the renamed region of interest and click Save. Make sure that the name specifies that the trace is for soma and save the file.To measure arborization area in the IBA1 channel, first select the Polygon Selection tool. Click on the microglial process extremity with the tool to start the polygon shape. Go around the microglia by clicking at the tips of each process extremity to form a polygon that best represents the area covered by the microglial arborizations.When the polygon is complete, click on the starting point to close it. Then, press the T key to add the trace to the ROI Manager. Select Analyze, Measure or press the M key to add the data to the Results window.Copy and paste this data to the data sheet. To save the information regarding the arborization area, go to the ROI Manager window, select the region of interest and change the name to match the image's name by pressing Rename. Right click on the renamed region of interest and click Save.Make sure that the name specifies that the trace is for arborization and save the file. This study described the step-by-step workflow for the immunofluorescent co-staining of IBA1 and TMEM119 and for the analysis of microglial density, distribution and morphology as well as of peripheral myeloid cell infiltration in mouse brain tissue. Fluorescence microscopy is used to image the co-labeling of microglia using IBA1 and TMEM119 in the coronal section of the dorsal hippocampus at 20X magnification.A successful staining reveals microglial cell bodies and their fine processes. The staining allows for the determination of microglial density and distribution and for the identification of microglial clusters and infiltrating macrophages. Confocal microscopy is used to image the stepwise microglial arborization tracing procedure at 40X magnification.These representative results show IBA1 positive and TMEM119 positive microglia as well as an example of cell body tracing. It is important to be consistent when counting cells and to pay attention to both signals of IBA1 and TMEM119. This technique allows the researcher to identify regions of the brain that are affected by a particular stimulus which can then be studied more in-depth with complementary techniques.

immunofluorescence-staining-using-iba1-tmem119-for-microglial-density

Research

JoVE Journal

Immunology and Infection

30.0K visualizações.  Centre de Recherche du CHU de Québec-Université Laval. Este protocolo mostrará como manchar microglia, realizar análises quantitativas de densidade e morfologia, bem como quantificar a infiltração de macrófagos no cérebro. Esta técnica permite ao pesquisador medir mudanças na distribuição e morfologia de microglia de forma quantitativa e diferenciar microglia de macrófagos infiltrados Este protocolo de análise pode ser facilmente aplicado a outros modelos animais para medir mudanças na distribuição e morfologia microglial onde anti...

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