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 saline (PBS) (Table 1). 
	NOTE: For optimal results, the brains should be perfused with 4% paraformaldehyde and cut to a thickness of 50 &#181;m with a vibratome. For a 24 multi-well plate, each well can hold up to six sections. The recommended volume of solution for each well is 350 &#181;L (for up to three sections) and 500 &#181;L for wells containing six sections. For a higher number of sections, it is recommended to use a 12 multi-well plate. Make sure that the selected volume of solution for each well completely covers the tissue and allows the sections to float. The recommended volumes apply for every solution used in the rest of the protocol.</li>
	<li>Wash the samples by covering them with 350 &#181;L of PBS and let them rest by placing the multi-well plate on top of a multipurpose shaker at room temperature (RT). Remove the PBS after 5 min and replace it 5x with fresh PBS. 
	NOTE: To remove the solutions, a transfer pipette is recommended. When pouring in any solution, make sure to place the tip of pipette against the well wall to protect tissue integrity. Also make sure to use a new pipette for each new solution.</li>
	<li>Remove PBS and add 350 &#181;L of 10 mM sodium citrate buffer with pH = 6.0 (Table 1).</li>
	<li>Seal the multi-well plate with paraffin film and let it float on a previously preheated water bath for 40 min at 70 &#176;C.</li>
	<li>Let the multi-well plate cool down for approximately 15 min.</li>
	<li>Remove the sodium citrate buffer and wash the sections in PBS as done in step 1.2.</li>
	<li>Remove PBS and add 350 &#181;L of freshly made 0.1% NaBH4 (Table 1) and let incubate for 30 min at RT.</li>
	<li>Remove the solution of 0.1% NaBH4 and wash the sections in PBS as done in step 1.2.</li>
	<li>Remove PBS and add blocking buffer (Table 1) for 1 h at RT on top of a multipurpose shaker. 
	NOTE: Make sure to prepare doubled volumes of blocking buffer, as the same solution will be used in the next step.</li>
	<li>Remove the blocking buffer and replace by blocking buffer containing the mixture of primary antibodies (1:150 mouse IBA1 + 1:300 TMEM119). Seal the plate with paraffin film and let it incubate overnight at 4 &#176;C.</li>
	<li>The next day, warm samples at RT for approximately 15 min.</li>
	<li>Wash the sections 5x for 5 min each in PBS with triton (PBST) (Table 1).</li>
	<li>Remove PBST and add blocking buffer containing the mixture of secondary antibodies (1:300 donkey anti-mouse Alexa 488 for IBA1; 1:300 goat anti-rabbit Alexa 568 for TMEM119) for 1.5 h at RT. Starting from this point onward, protect the samples from light.</li>
	<li>Remove blocking buffer and wash the sections 5x as done in step 1.2, except this time with PBST.</li>
	<li>Remove the PBST and add 4&#8242;,6-diamidino-2-phenylindole (DAPI) [1:20000] for 5 min at RT.</li>
	<li>Remove DAPI and wash the sections 3x for 5 min each in phosphate buffer (PB).</li>
	<li>Mount the sections on a microscope slide. Let them dry while protected from light.</li>
	<li>When dried, add some drops of mounting fluorescence medium and cover with a coverslip, avoiding bubble formation. 
	NOTE: Store the slides while protected from light, inside a histological slide box, at 4 &#176;C. The samples can be preserved for several months.</li>
</ol>
2. Imaging for density and distribution analysis
<ol>
	<li>With the help of a widefield epifluorescence microscope, use a low magnification and the DAPI channel to locate the ROI (i.e., the CA1 region of the hippocampus).</li>
	<li>Acquire images at 20x, using a numerical aperture (NA) of 0.5, with the DAPI, 488, and 568 channels and filters, at a resolution of 0.3 &#181;m/pixel. Capture a mosaic picture covering the ROI. Alternatively, take individual pictures that will be stitched into a larger image. 
	NOTE: A mosaic image is a super image constituted by smaller images. Mosaic images are usually used to overcome the limited area of the field-of-view of high magnifications. Some software includes a mosaic function; nevertheless, images can also be manually stitched together with other photo editing software by stitching the individual images into one. Remember to add the scale information to the file. For this type of analysis, it is recommended to have at least 300 microglial cells imaged per ROI/animal (corresponding to approximately 10&#8722;15 pictures for the hippocampus, for example), with a minimum of five animals per experimental condition. Figure 1A&#8722;C shows the images of colabelled microglia.</li>
	<li>Save the image as a TIFF file.</li>
</ol>
3. Imaging for morphology analysis
<ol>
	<li>Using a confocal or structured illumination microscope, use the DAPI channel to locate the ROI at low magnification.</li>
	<li>Using a 40x objective (i.e., NA 1.4 oil), locate an IBA1+/TMEM119+ cell inside the ROI. While live imaging, move in the Z-axis. As soon as the signal of the randomly selected microglia disappears, set this Z-level as the beginning of the Z-stack. Move along the Z-axis in the opposite direction until the signal of the microglia disappears and set that point as the end of the Z-stack. 
	NOTE: Figure 2A&#8722;C shows images of IBA1+/TMEM119+ microglia.</li>
	<li>Create a Z-stack in all three channels (DAPI, 488, 568) using a 0.33 &#181;m Z-interval and pixel size of 0.15 &#181;m/pixel. Add the scale information to the file. 
	NOTE: The recommended Z-interval depends on the resolving power of the objective (e.g., for a 40x objective such as NA 1.4 oil, it is 0.33 &#181;m). For morphology analysis, it is recommended to have at least 20 cells per animal with a minimum of five animals per experimental condition.</li>
	<li>Save the file as a TIFF file.</li>
</ol>
4. Density and distribution analysis
<ol>
	<li>Open FIJI/ImageJ with the nearest neighbor distance (NND) plugin installed. Open the 20x image. 
	NOTE: Use a search engine with the keyword &#8220;Nearest Neighbor Distances Calculation with ImageJ&#8221; to find the installation instructions. The plugin Author is Yuxiong Mao.</li>
	<li>To set the scale manually based on a scale imprinted on the image, select the straight line tool (Figure 3E), 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 (Figure 3I), select Analyze | Set scale, then enter the correct information (Figure 3J). 
	NOTE: The scale can sometimes be contained in the metadata of the file and set automatically.</li>
	<li>Select Image | Color | Make composite to create a composite image of all channels. 
	NOTE: During image acquisition, FIJI/ImageJ will automatically create a composite in the RGB format.</li>
	<li>On the menu bar, select Analyze | Set measurements. Check Area, Centroid, and Perimeter. On the tab Redirect to, click and select the opened file (Figure 3K).</li>
	<li>Go to Image | Color | Channel tool to open the channel tool. 
	NOTE: This menu will allow a specific color to be disabled. The DAPI channel can be useful to identify ROI and to confirm cells. It can be deactivated to make counting easier.</li>
	<li>Draw a rough perimeter of the ROI with the freehand selection tool (Figure 3D).</li>
	<li>Enable the selection brush tool by double-clicking the oval tool on the tool bar and make sure that the Enable selection brush box is checked (Figure 3G). This tool will be used to delineate the ROI more precisely. Select an appropriate brush size between 200&#8722;400.</li>
	<li>Using the selection brush, adjust the perimeter to best fit the ROI. Press T on the keyboard to add to the ROI manager (Figure 3L).</li>
	<li>Select Analyze | Measure or press the M key, and a results window will pop up. Copy and paste the results on a datasheet, then save the information regarding the area (i.e., the area of the ROI; Figure 3R).</li>
	<li>After copying the area of the ROI, erase the information from the results window by clicking on it and pressing the Backspace key.</li>
	<li>Go to the ROI manager window (Figure 3L), right-click the ROI trace, change the name to match the image&#8217;s name, then save.</li>
	<li>Double-click the brush tool at the tool bar. Select the black color and a brush size of 10. Make sure that the option Paint of overlay is unchecked (Figure 3H).</li>
	<li>In the TMEM119 channel, carefully place a black dot on the center the soma for each TMEM119+ microglia. Place a white dot on the center of the cells that are not positive for TMEM119 (to mark infiltrating macrophages). Repeat the same procedure for all cells contained in the ROI. 
	NOTE: It is important that all dots (black and white) are located in the same channel. The identity of the channel can be verified (red, blue, or green) by looking at the color of the image window labels.</li>
	<li>Select Image | Color | Split channel. A window for each channel will appear. Then, identify the channel that has the dot annotations and close the other two windows.</li>
	<li>Redirect the new split channel image. Go to Analyze | Set measurements. On the tab Redirect to, click and select the split channel image (Figure 3K).</li>
	<li>Select Image | Type | 8-bit. Go to Image | Adjust and select Threshold (Figure 3O). To adjust the threshold, slide the button of the second bar, all the way to the left (threshold value = 0) in both bars. 
	NOTE: This will leave only the black dots on the image, appearing white.</li>
	<li>Select the ROI in the ROI manager window. Select Analyze | Analyze particle (Figure 3N). On Size (inch&#710;2): write 1&#8722;20. Keep the pixel unit unchecked, check Display, summarize and add to manager, and press Ok. The summary window will pop up and give the number of points (Figure 3P). Copy and paste the information to the datasheet.</li>
	<li>Select Plugins | NND. The NND window will pop up (Figure 3Q). Copy/paste all the information to the datasheet. Each number represents the distance each microglia has to the nearest neighboring microglia.</li>
	<li>Go back to the threshold window and slide the first bar all the way to the right (threshold value = 255 in both bars), which will leave all the white dots visible, appearing white (Figure 3M).</li>
	<li>Select Analyze | Analyze particle. The summary window that provides the number of points will pop up (Figure 3P). Copy and paste the information to the datasheet.</li>
	<li>Go to the ROI manager select all the points, right-click, and save with the image&#8217;s name. This will allow saving of all the points in a zip file (Figure 3L). Select File | Save as, and save the file with a name that allows identification of the analyzed image.</li>
	<li>Obtain the density of microglia (for each image) by dividing the number of IBA1+/TMEM119+ double-positive cells by the area of the ROI. 
	NOTE: The values for each picture can be averaged for each animal. The data can then be presented as mean &#177; standard error of the mean (SEM) of all the animals.</li>
	<li>Determine the NND by obtaining an average per picture of the NND values of all TMEM119+ cells. 
	NOTE: The data can then be presented as mean &#177; SEM of all the animals.</li>
	<li>Calculate the spacing index using the formula: NND2 x density. 
	NOTE: The data can then be presented as mean &#177; SEM of all the animals. The units for this measurement will be arbitrary units.</li>
	<li>Quantify microglial clusters by identifying cells that have an NND under 12 &#181;m. 
	NOTE: Here, 12 &#181;m is selected, as it is the approximate distance between two directly juxtaposing microglial cells touching each another with arborizations. If there are more than three microglia that meet this condition, return to the image and verify whether these cells are part of one or multiple clusters.</li>
	<li>After confirming the number of clusters, write the number of clusters in the datasheet. 
	NOTE: The number of clusters can be divided by the ROI area to obtain the density of cells/mm2 for each animal. The data can then be presented as mean &#177; SEM of all the animals.</li>
	<li>To determine the percentage of peripheral myeloid cell infiltration, calculate the % of IBA1+/TMEM119- cells over the total number of myeloid cells (TMEM119+/IBA1+ + TMEM119-/IBA1+) for each animal. 
	NOTE: The data can then be presented as mean &#177; SEM of all the animals.</li>
</ol>
5. Morphology analysis
<ol>
	<li>Open FIJI/ImageJ.</li>
	<li>Open the 40x image using Image J or FIJI. Select A popup window will appear asking if the images should be opened in a stack. Click OK. Next, select Image | Stacks | Z project 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 </li>
	<li>Click on the new window with the Z project. Select Image | Colors | Split channels. Conduct the traces on the images of the IBA1 channel. 
	NOTE: The other channels (TMEM119 and DAPI) can be kept open and consulted as needed during the microglial morphology analysis.</li>
	<li>On the menu bar, select Analyze | Set measurements. Check the Area, Centroid, and Perimeter. On the tab Redirect to, select the opened file (Figure 3K).</li>
	<li>Set the scale as described in steps 4.2.</li>
	<li>To measure the soma size in the IBA1 channel, draw a rough perimeter of the soma with the freehand selection tool (Figure 3D).</li>
	<li>Enable the selection brush tool by double-clicking the oval tool on the tool bar, followed by checking Enable the selection brush box (Figure 3G). Select a selection brush size between 10&#8722;20 (Figure 3B).</li>
	<li>Using the selection brush, adjust the trace to best fit the soma. Zooming in will enable precision during this step (Figure 2I).</li>
	<li>Press the T key to add the soma trace to the ROI manager (Figure 3L).</li>
	<li>Select Analyze | Measure or press the M key. A results window will pop up. Copy and paste the results on a datasheet (Figure 3R).</li>
	<li>To save the information regarding the soma area, go to the ROI manager window, right-click on the ROI, change the name to match the image&#8217;s name, specify that the trace is for soma, then save the file.</li>
	<li>To measure arborization area in the IBA1 channel, click on a microglial process extremity with the polygon selection tool, which will start the polygon shape (Figure 3C).</li>
	<li>Following the tips of the microglial processes, 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 (Figure 2D&#8722;H). 
	NOTE: Make sure that the polygon connects all the microglial process extremities. The lines forming the polygon should never intersect. When clicking around a microglial process tip, be careful to avoid cutting off any part of the process. It is sometimes useful to add extra points to go around a process. The number of points forming the polygon is not directly linked to the number of distal processes and thus is not relevant for the study.</li>
	<li>To close the polygon, click on the starting point of the polygon.</li>
	<li>Press the T key to add the trace to the ROI manager (Figure 3L). Select Analyze | Measure or press the M key. A results window will pop up. Copy and paste the results on a datasheet (Figure 3R).</li>
	<li>To save the information regarding the arborization area, go to the ROI manager window, right-click the ROI, change the name to match the image&#8217;s name, specify for arborization, then save the file.</li>
	<li>Determine the soma area by averaging all soma areas for each animal. 
	NOTE: The data can be presented as mean &#177; SEM of all the animals.</li>
	<li>Determine arborization area by averaging all the arborization areas for each animal. 
	NOTE: The data can be presented as mean &#177; SEM of all the animals.</li>
	<li>Calculate the morphology index by using the formula soma area/arborization area for each microglial cell and average per animal. 
	NOTE: The data can be presented as mean &#177; SEM of all the animals.</li>
</ol>

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The authors have nothing to disclose.

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><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>Na&lt;sub&gt;2&lt;/sub&gt;H&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;H&lt;sub&gt;2&lt;/sub&gt;O</td><td>BioShop Canada Inc.</td><td>SPM306, SPM400</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>BioShop Canada Inc.</td><td>SPD307, SPD600</td><td></td></tr><tr><td>NaBH&lt;sub&gt;4&lt;/sub&gt;</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−C). This staining allows determination of microglial density and distribution and identification of microglial clusters (Figure 1I<...

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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 ...

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

Research

JoVE Journal

Immunology and Infection

31.7K Views.  Centre de Recherche du CHU de Québec-Université Laval. Dit protocol zal u laten zien hoe microglia vlek, uitvoeren van kwantitatieve dichtheid en morfologie analyse, alsmede om macrofaag infiltratie in de hersenen te kwantificeren. Deze techniek stelt de onderzoeker in staat om veranderingen in microgliadistributie en morfologie op een kwantitatieve manier te meten en microglia te onderscheiden van het infiltreren van macrofagen Dit analyseprotocol kan eenvoudig worden toegepast op andere diermodellen om veranderingen in microgliadistributie en m...

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

Preparation of Mouse Brain Tissue for Immunoelectron Microscopy

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments (dendrite, dendritic spine, axon, axon terminal, perikaryon), as well as their intracellular organelles and cytoskeleton, in the central nervous system at high spatial resolution. Combined with TEM, pre-embedding immunocytochemistry allows the discrimination of cellular elements with few distinctive features and identification criteria (e.g., microglial perikarya and processes, when using an antibody against the microglia-specific marker Iba1 (ionized calcium binding adaptor molecule 1; as presented here)), identifying the neurotransmitter contents of cellular elements (e.g., serotonergic) and their ultrastructural localization of soluble or membrane-bound proteins (e.g., 5 HT1A and EphA4 receptors). Here, we describe a protocol for transcardiac perfusion of mice with acrolein fixative, removal and sectioning of the brain, as well as immunoperoxidase-diaminobenzidine (DAB) staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with TEM. When rigorously performed, this technique provides an excellent compromise between optimal ultrastructural preservation and immunocytochemical detection.

We describe a protocol for transcardiac perfusion of mice, removal and sectioning of the brain, as well as immunoperoxidase staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with transmission electron microscopy.

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments ...

Neuroscience

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury

After brain stroke microglia/macrophages (M/M) undergo rapid activation with dramatic morphological and phenotypic changes that include expression of novel surface antigens and production of mediators that build up and maintain the inflammatory response. Emerging evidence indicates that M/M are highly plastic cells that can assume classic pro-inflammatory (M1) or alternative anti-inflammatory (M2) activation after acute brain injury. However a complete characterization of M/M phenotype marker expression, their colocalization and temporal evolution in the injured brain is still missing.	
	Immunofluorescence protocols specifically staining relevant markers of M/M activation can be performed in the ischemic brain. Here we present immunofluorescence-based protocols followed by three-dimensional confocal analysis as a powerful approach to investigate the pattern of localization and co-expression of M/M phenotype markers such as CD11b, CD68, Ym1, in mouse model of focal ischemia induced by permanent occlusion of the middle cerebral artery (pMCAO). Two-dimensional analysis of the stained area reveals that each marker is associated to a defined M/M morphology and has a given localization in the ischemic lesion. Patterns of M/M phenotype marker co-expression can be assessed by three-dimensional confocal imaging in the ischemic area. Images can be acquired over a defined volume (10 &mu;m z-axis and a 0.23 &mu;m step size, corresponding to a 180 x 135 x 10 &mu;m volume) with a sequential scanning mode to minimize bleed-through effects and avoid wavelength overlapping. Images are then processed to obtain three-dimensional renderings by means of Imaris software. Solid view of three dimensional renderings allows the definition of marker expression in clusters of cells. We show that M/M have the ability to differentiate towards a multitude of phenotypes, depending on the location in the lesion site and time after injury.

A way to gain new insights into the complexity of the brain inflammatory response is presented. We describe immunofluorescence-based protocols followed by three-dimensional confocal analysis to investigate the pattern of co-expression of microglia/macrophage phenotype markers in a mouse model of focal ischemia.

After brain stroke microglia/macrophages (M/M) undergo  rapid activation with dramatic morphological and phenotypic changes that  include expression of ...

Antibody Binding Specificity for Kappa (V&#954;) Light Chain-containing Human (IgM) Antibodies: Polysialic Acid (PSA) Attached to NCAM as a Case Study

Antibodies of the IgM isotype are often neglected as potential therapeutics in human trials, animal models of human diseases as well as detecting agents in standard laboratory techniques. In contrast, several human IgMs demonstrated proof of efficacy in cancer models and models of CNS disorders including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). Reasons for their lack of consideration include difficulties to express, purify and stabilize IgM antibodies, challenge to identify (non-protein) antigens, low affinity binding and fundamental knowledge gaps in carbohydrate and lipid research. 
This manuscript uses HIgM12 as an example to provide a detailed protocol to detect antigens by Western blotting, immunoprecipitations and immunocytochemistry. HIgM12 targets polysialic acid (PSA) attached to the neural cell adhesion molecule (NCAM). Early postnatal mouse brain tissue from wild type (WT) and NCAM knockout (KO) mice lacking the three major central nervous system (CNS) splice variants NCAM180, 140 and 120 was used to evaluate the importance of NCAM for binding to HIgM12. Further enzymatic digestion of CNS tissue and cultured CNS cells using endoneuraminidases led us to identify PSA as the specific binding epitope for HIgM12.

We present a protocol to identify protein moieties containing antigens for human and mouse IgM antibodies with specific kappa (V&#954;) light chains. This protocol is not limited to IgM class antibodies but applies to all immunoglobulin isotypes that target their antigen with sufficiently high affinity during immunoprecipitations.

Antibodies of the IgM isotype are often neglected as potential therapeutics in human trials, animal models of human diseases as well as detecting agents ...

Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brain tissue, using markers for microglia and neuronal elements as an example. Specifically, this paper is a step-by-step protocol for fluorescent visualization of microglia and neurons via immunohistochemistry for Iba1 and Pan-neuronal, respectively. Fluorescence double-labeling is particularly useful for the localization of multiple proteins within the same sample, providing the opportunity to accurately observe interactions between cell types, receptors, ligands, and/or the extracellular matrix in relation to one another as well as protein co-localization within a single cell. Unlike other visualization techniques, fluorescence immunohistochemistry staining intensity may decrease in the weeks to months following staining, unless appropriate precautions are taken. Despite this limitation, in many applications fluorescence double-labeling is preferred over alternatives such as 3,3&#39;-diaminobenzidine tetrahydrochloride (DAB) or alkaline phosphatase (AP), as fluorescence is more time efficient and allows for more precise differentiation between two or more markers. The discussion includes troubleshooting tips and advice to promote success.

This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brains, using markers for microglia and neuronal elements as an example.

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level ...

Correlative Light and Electron Microscopy to Study Microglial Interactions with &#946;-Amyloid Plaques

A detailed protocol is provided here to identify amyloid A&#946; plaques in brain sections from Alzheimer&#39;s disease mouse models before pre-embedding immunostaining (specifically for ionized calcium-binding adapter molecule 1 (IBA1), a calcium binding protein expressed by microglia) and tissue processing for electron microscopy (EM). Methoxy-X04 is a fluorescent dye that crosses the blood-brain barrier and selectively binds to &#946;-pleated sheets found in dense core A&#946; plaques. Injection of the animals with methoxy-X04 prior to sacrifice and brain fixation allows pre-screening and selection of the plaque-containing brain sections for further processing with time-consuming manipulations. This is particularly helpful when studying early AD pathology within specific brain regions or layers that may contain very few plaques, present in only a small fraction of the sections. Post-mortem processing of tissue sections with Congo Red, Thioflavin S, and Thioflavin T (or even with methoxy-X04) can label &#946;-pleated sheets, but requires extensive clearing with ethanol to remove excess dye and these procedures are incompatible with ultrastructural preservation. It would also be inefficient to perform labeling for A&#946; (and other cellular markers such as IBA1) on all brain sections from the regions of interest, only to yield a small fraction containing A&#946; plaques at the right location. Importantly, A&#946; plaques are still visible after tissue processing for EM, allowing for a precise identification of the areas (generally down to a few square millimeters) to examine with the electron microscope.

This article describes a protocol for visualizing amyloid A&#946; plaques in Alzheimer&#39;s disease mouse models using methoxy-X04, which crosses the blood-brain barrier and selectively binds to &#946;-pleated sheets found in dense core A&#946; plaques. It allows pre-screening of plaque-containing tissue sections prior to immunostaining and processing for electron microscopy.

A detailed protocol is provided here to identify amyloid A&#946; plaques in brain sections from Alzheimer&#39;s disease mouse models before pre-embedding ...

Quantifying Microglia Morphology from Photomicrographs of Immunohistochemistry Prepared Tissue Using ImageJ

Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. As the first responders, microglia have important functions to mitigate neuron and glia dysfunction, and in this process, they undergo a broad range of morphologic changes. Microglia morphologies can be categorized descriptively or, alternatively, can be quantified as a continuous variable for parameters such as cell ramification, complexity, and shape. While methods for quantifying microglia are applied to single cells, few techniques apply to multiple microglia in an entire photomicrograph. The purpose of this method is to quantify multiple and single cells using readily available ImageJ protocols. This protocol is a summary of the steps and ImageJ plugins recommended to convert fluorescence and bright-field photomicrographs into representative binary and skeletonized images and to analyze them using software plugins AnalyzeSkeleton (2D/3D) and FracLac for morphology data collection. The outputs of these plugins summarize cell morphology in terms of process endpoints, junctions, and length as well as complexity, cell shape, and size descriptors. The skeleton analysis protocol described herein is well suited for a regional analysis of multiple microglia within an entire photomicrograph or region of interest (ROI) whereas FracLac provides a complementary individual cell analysis. Combined, the protocol provides an objective, sensitive, and comprehensive assessment tool that can be used to stratify between diverse microglia morphologies present in the healthy and injured brain.

Microglia are brain immune cells that survey and react to altered brain physiology through morphologic changes which may be evaluated quantitatively. This protocol outlines an ImageJ based analysis protocol to represent microglia morphology as continuous data according to metrics such as cell ramification, complexity, and shape.

Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. As ...

Delivery of Antibodies into the Brain Using Focused Scanning Ultrasound

Only a small fraction of therapeutic antibodies targeting brain diseases are taken up by the brain. Focused ultrasound offers a possibility to increase uptake of antibodies and engagement through transient opening of the blood-brain barrier (BBB). In our laboratory, we are developing therapeutic approaches for neurodegenerative diseases in which an antibody in various formats is delivered across the BBB using microbubbles, concomitant with focused ultrasound application through the skull targeting multiple spots, an approach we refer to as scanning ultrasound (SUS). The mechanical effects of microbubbles and ultrasound on blood vessels increases paracellular transport across the BBB by transiently separating tight junctions and enhances vesicle- mediated transcytosis, allowing antibodies and therapeutic agents to effectively cross. Moreover, ultrasound also facilitates the uptake of antibodies from the interstitial brain into brain cells such as neurons where the antibody distributes throughout the cell body and even into neuritic processes. In our studies, fluorescently labeled antibodies are prepared, mixed with in-house prepared lipid-based microbubbles and injected into mice immediately before SUS is applied to the brain. The increased antibody concentration in the brain is then quantified. To account for alterations in normal brain homeostasis, microglial phagocytosis can be used as a cellular marker. The generated data suggest that ultrasound delivery of antibodies is an attractive approach to treat neurodegenerative diseases.

Presented here is a protocol to transiently open the blood-brain barrier (BBB) either focally or throughout a mouse brain to deliver fluorescently-labeled antibodies and activate microglia. Also presented is a method to detect the delivery of antibodies and microglia activation by histology.

Only a small fraction of therapeutic antibodies targeting brain diseases are taken up by the brain. Focused ultrasound offers a possibility to increase ...

Transplantation of Human Induced Pluripotent Stem Cell-Derived Microglia in Immunocompetent Mice Brain via Non-Invasive Transnasal Route

Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain functions. Most of our current understanding of microglia is based on experiments performed in the mouse. Human microglia differ from mouse microglia, and thus response and characteristics of mouse microglia may not always represent that of human microglia. Further, due to ethical and technical difficulties, research on human microglia is restricted to in vitro culture system, which does not capitulate in vivo characteristics of microglia. To overcome these issues, a simplified method to non-invasively transplant induced pluripotent stem cell-derived human microglia (iPSMG) into the immunocompetent mice brain via a transnasal route in combination with pharmacological depletion of endogenous microglia using a colony-stimulating factor 1 receptor (CSF1R) antagonist is developed. This protocol provides a way to non-invasively transplant cells into the mouse brain and may therefore be valuable for evaluating the in vivo role of human microglia in physiological and pathological brain functions.

The protocol presented here allows the transplantation of induced pluripotent stem cell-derived human microglia (iPSMG) into the brain via a transnasal route in immunocompetent mice. The method for the preparation and transnasal transplantation of cells and the administration of cytokine mixture for the maintenance of iPSMG is shown.

Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain ...

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

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