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

This protocol will show you how to stain microglia, perform quantitative density and morphology analysis as well as to quantify macrophage infiltration into the brain. This technique allows the researcher to measure changes in microglia distribution and morphology in a quantitative way and to differentiate microglia from infiltrating macrophages This analysis protocol can be easily applied to other animal models to measure changes in microglial distribution and morphology where anti-IBA1 and anti-TMEM119 antibodies are available. It is highly recommended to conduct the cell counts and traces blinded to the experimental condition and to be consistent in the way this procedure is performed.

To begin this procedure, open an image processing program such Fiji or ImageJ that has the nearest neighbor distance plug-in installed. Open the 20X image if the scale is contained in the metadata of the file and not automatically set from the metadata. On the menu bar, select Analyze, Set Scale, then enter the correct information.

To set the scale manually based on a scale imprinted on the image, select the Straight Line tool. Place the cursor on the edge of the scale and while pressing the Shift key, draw a line as close as possible to the scale on the image. Select Analyze, Set Scale.

A window will pop up and put the correct known distance and unit of length to determine the pixel's per length unit and click OK.Next, select Image, Color, Make Composite to create a composite image of all channels. On the menu bar, select Analyze, Set Measurements. Check Area, Centroid and Perimeter.

In the Redirect To dropdown menu, select the Open file then click OK.Go to Image, Color, Channel Tool to open the Channel tool. Use the Freehand Selection tool to draw a rough perimeter around the region of interest. Double click the Oval tool on the tool bar to enable the selection Brush tool.

Make sure that the Enable Selection Brush box is checked and click OK.Using the selection brush, adjust the perimeter to best fit the region of interest, then press T on the keyboard throughout this area to arrow Y manager. Select Analyze, Measure or Press the M key and the Results window will pop up. Copy and paste the results into a data sheet and save the information regarding the area.

After copying the area of the region of interest, you base the information from the Results window by clicking on it and pressing the Backspace key. Select the ROI Manager window, select the ROI Trace and change the name to match the image's name by pressing Rename. Click OK then right click the new name of the ROI Trace and save.

Double click the Brush tool in the tool bar. Select the color black and the Brush size of 10. Make sure that the option Paint on overlay is unchecked.

In the TMEM119 channel, carefully place a black dot on the center of the soma for each TMEM119 positive microglia. Place a white dot on the center of the cells that are not positive for TMEM119. Repeat the same procedure for all cells contained in the region of interest.

Next, select Image, Color, Split Channel, a window for each channel will appear. Identify the channel that has the dot annotations and close the other two windows. To redirect the new split channel image, go to Analyze, Set Measurements.

In the Redirect To drop down menu, select the Split Channel Image and click OK.Then select Image, Type, eight-bit. Go to Image Adjust and select Threshold. Adjust the sliders all the way to the left in both bars.

This will leave only the black dots which will now appear white. Select the region of interest in the ROI Manager window. In the Menu bar, select Analyze, Analyze Particle.

In the text box for Size, input one through 20. The box for Pixel units should remain unchecked while the box that's for Display results, Summarize and Add to Manager should be checked. Click OK to display the Summary window which will give the number of points.

Copy and paste this information to the data sheet. Then, select plug-ins NND to show the NND window. Each number represents the distance each microglia has to the nearest neighboring microglia.

Copy and paste all of this information to the data sheet. Go back to the Threshold window and adjust the top slider all the way to the right, which will leave all of the white dots visible, now appearing white. Select Analyze, Analyze Particle to display the Analyze Particles pop up window.

Click OK to show the Summary window that provides the number of points. Copy and paste this information into the data sheet. Go to the ROI Manager and select all the points.

Then right click and Save with the image's name. This will allow saving of all the points in a ZIP file. First, open the 4DX image files.

A pop up window will appear asking if the images should be opened in a stack. Click OK.Next, select Image, Stacks, ZProject to open the Z Projection window. Include all slices from the first through last slice.

Ensure that the Max Intensity is selected under Projection Type and click OK.Click on the new window that contains the ZProject. Select Image, Colors, Split Channels and conduct the traces on the images of the IBA1 channel. In the Menu bar, select Analyze, Set Measurements.

Check the Area, Centroid and Perimeter. In the Redirect To drop down menu, select the open file and click OK.To set the scale manually based on a scale imprinted on the image, select the Straight Line tool. Place the cursor on the edge of the scale and while pressing the Shift key, draw a line as close as possible to the scale on the image.

Select Analyze, Set Scale. A window will pop up. Input the correct known distance and unit of length to determine the Pixels per Length unit and click OK.To measure the soma size in the IBA1 channel, draw a rough perimeter of the soma with the Freehand Selection tool.

Double click the Oval tool on the toolbar to enable the Selection Brush tool. Make sure that the Enable Selection Brush box is checked and click OK.Using the Selection Brush, adjust the trace to best fit the soma. Zooming in will enable precision during this step.

Next, press the T key to add the soma trace to the ROI Manager. Select Analyze, Measure or press the M key to display the results. Copy and paste these results to a data sheet.

Go to the ROI Manager window, select the region of interest and change the name to match the image's name by pressing Rename. Right click on the renamed region of interest and click Save. Make sure that the name specifies that the trace is for soma and save the file.

To measure arborization area in the IBA1 channel, first select the Polygon Selection tool. Click on the microglial process extremity with the tool to start the polygon shape. Go around the microglia by clicking at the tips of each process extremity to form a polygon that best represents the area covered by the microglial arborizations.

When the polygon is complete, click on the starting point to close it. Then, press the T key to add the trace to the ROI Manager. Select Analyze, Measure or press the M key to add the data to the Results window.

Copy and paste this data to the data sheet. To save the information regarding the arborization area, go to the ROI Manager window, select the region of interest and change the name to match the image's name by pressing Rename. Right click on the renamed region of interest and click Save.

Make sure that the name specifies that the trace is for arborization and save the file. This study described the step-by-step workflow for the immunofluorescent co-staining of IBA1 and TMEM119 and for the analysis of microglial density, distribution and morphology as well as of peripheral myeloid cell infiltration in mouse brain tissue. Fluorescence microscopy is used to image the co-labeling of microglia using IBA1 and TMEM119 in the coronal section of the dorsal hippocampus at 20X magnification.

A successful staining reveals microglial cell bodies and their fine processes. The staining allows for the determination of microglial density and distribution and for the identification of microglial clusters and infiltrating macrophages. Confocal microscopy is used to image the stepwise microglial arborization tracing procedure at 40X magnification.

These representative results show IBA1 positive and TMEM119 positive microglia as well as an example of cell body tracing. It is important to be consistent when counting cells and to pay attention to both signals of IBA1 and TMEM119. This technique allows the researcher to identify regions of the brain that are affected by a particular stimulus which can then be studied more in-depth with complementary techniques.