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 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...

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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

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Immunology and Infection

31.3K 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

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and epidermis home a variety of immune cells in both homeostatic and inflammatory conditions. Tools to obtain skin cell release for cytofluorimetric analyses are, therefore, very useful in order to study the complex network of immune cells residing in the skin and their response to microbial stimuli. Here, we describe an efficient methodology for the digestion of mouse skin to rapidly and efficiently obtain single-cell suspensions. This protocol allows maintenance of maximum cell viability without compromising surface antigen expression. We also describe how to take and digest skin samples from different anatomical locations, such as the ear, trunk, tail, and footpad. The obtained suspensions are then stained and analyzed by flow cytometry to discriminate between different leukocyte populations.

The skin is home to a complex immune cell network. We describe an efficient methodology for the digestion of mouse skin, from different parts of the animal's body, in order to obtain a single-cell suspension and analyze the different leukocyte populations resident in the skin by flow cytometry.

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain insight into an ongoing disease state.
Here, a simple and efficient method is described to perform BAL on murine lungs without the need of special tools or equipment. BAL fluid is isolated by inserting a catheter in the trachea of terminally anesthetized mice, through which a saline solution is instilled into the bronchioles. The instilled fluid is gently retracted to maximize BAL fluid retrieval and to minimize shearing forces. This technique allows the viability, function, and structure of cells within the airways and BAL fluid to be preserved.
Numerous techniques may be applied to gain further understanding of the disease state of the lung. Here, a commonly used technique for the identification and enumeration of different types of immune cells is described, where&#160;flow cytometry is combined with a select panel of fluorescently labeled cell surface-specific markers. The BAL procedure presented here can also be used to analyze infectious agents, fluid constituents, or inhaled particles within murine lungs.

The health status of the lung is reflected by the type and number of immune cells that are present in the bronchioles of the lung. We describe a bronchoalveolar lavage technique that allows the isolation and study of nonadherent cells and soluble factors from the lower respiratory tract of mice.

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain ...

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...

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

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