Analysis of Astrocyte Territory Volume and Tiling in Thick Free-Floating Tissue Sections

This protocol describes methods for sectioning, staining, and imaging free-floating tissue sections of the mouse brain, followed by a detailed description of the analysis of astrocyte territory volume and astrocyte territory overlap or tiling.

Understanding how astrocytes develop and establish their complex morphology is essential to understanding how the brain develops and functions. This protocol describes how to analyze key aspects of astrocyte morphology in brain tissue. This protocol uses a custom code to measure astrocyte territory volume in thick tissue sections.

This strategy can also be applied to measure the amount of territory overlap between neighboring astrocytes. By combining this protocol with genetic manipulation of astrocytes, researchers can directly investigate the role of specific proteins and pathways in astrocyte development and astrocyte-astrocyte interaction To begin, prepare a fresh solution of TBST by adding 1 milliliter of 10%Triton X to a 50-milliliter tube and filling the tube to 50 milliliters with TBS. Prepare 2 milliliters of blocking and antibodies solutions for each brain by combining goat serum and TBST in a 15-milliliter tube.

Next, label a 24-well plate to place different samples in different rows in different solutions in different columns, then add one milliliter of TBST to the first three columns labeled as wash 1, wash 2, and wash 3, and add 1 milliliter of blocking solution to the fourth column. Prepare a glass pick by melting the end of a 5.75-inches Pasteur pipette into a small hook using a Bunsen burner, then transfer tissue sections from the 12-well plate into the wash 1 column of the 24-well plate using the pick, then wash the sections for 10 minutes each in wash 1, 2, and 3 wells by using the glass pick to transfer the sections from one well to the next, followed by incubating the sections for one hour in the blocking solution. Prepare q milliliter of primary antibody solution by adding the antibody to the antibody solution, then vortex briefly and centrifuge for 5 minutes at greater than or equal to 4, 000 times g at 4 degrees Celsius.

Next, incubate the sections in primary antibody for 2 to 3 nights at 4 degrees Celsius while shaking. After primary antibody incubation, aspirate the day-1 TBST from the wash wells, then add 1 milliliter of new TBST to each wash well and move the sections into the first wash well. Next, incubate sections in secondary antibody for 3 hours at room temperature.

After secondary antibody incubation, aspirate the day-2 TBST from the wash wells, then add 1 milliliter of new TBST to each wash well and move the sections into the first wash well. During the final wash, take the mounting media out from 4 degrees Celsius and allow it to warm to room temperature, then prepare a 2-to-1 mixture of TBS and deionized water and add it to a Petri dish. Next, prepare a microscope slide by adding 800 microliters of 2-to-1 mixture of TBS and deionized water to the surface.

Transfer the sections from the wash 3 well to the Petri dish, then using a fine paintbrush, transfer the sections one at a time from the Petri dish into the liquid on the slide. Next, carefully arrange the sections so that they are flat on the slide using the paintbrush. Carefully, remove excess liquid from the slide with a P-1000 pipette, followed by vacuum aspiration.

Once all excess liquid is removed from the slide, use a transfer pipette to immediately add 1 drop of mounting media to each section and gently lay a cover slip over the slide. Allow mounting media to spread for a few minutes and then remove any excess mounting media that comes out from under the cover slip by vacuum aspiration. Afterward, seal all four edges of the cover slip with clear nail polish.

Let slides dry for 30 minutes at room temperature and then store them flat at 4 degrees Celsius. Using a 10x objective, locate individual cells for imaging and note the specific brain region or subregion relevant to the experiment, then using the focus knob, determine whether the entire astrocyte is contained within the section. Afterward, switch to a higher magnification oil objective and bring the cell into focus.

While looking through the eye piece, use the focus knob to move from the top to the bottom of the cell, then visually inspect the cell to ensure that the entire cell is contained within the section. Using the confocal microscopes associated acquisition software, adjust the acquisition parameters to obtain an appropriate signal-to-noise ratio and level of detail, then use 2x zoom for a 40x objective and no zoom if using a 60x or a 63x objective. Set the upper and lower boundaries of the Z-stack with a step size of 0.5 micrometers ensuring that the entire astrocyte is being captured with the first and last images without any fluorescent labeling of the cell.

Before beginning analysis, install the convex hull extension file, XtspotsConvexHull, by pasting the file in the rtmatlab sub-folder of the XT folder. Using the Imaris File Converter, convert images to IMS format. Next, open an image file in the image analysis software and select the 3D View button to view the image in the 3D mode, ensuring that the cell meets the inclusion criteria.

To create a surface, click on the blue Add New Surfaces icon, then create a region of interest around the cell by entering dimensions into the boxes under size of the surface creation tool or dragging the edges of the yellow box. Next, adjust the Threshold Absolute Intensity by dragging the yellow bar or inputting values in the box so that the gray surface fills as much of the cell signal as possible without going beyond the boundary of the cell. Afterward, click on the green arrow to finish generating the surface.

Carefully inspect the newly generated surface and delete any surface pieces that are not part of the cell by selecting them, then clicking on the Pencil Edit tool followed by the Delete option. To unify the remaining surface pieces, click on the Funnel Filter icon to select all, then click on the Pencil Edit icon and select the Unify option. Click on the orange Add New Spots icon and select the correct source channel from the drop-down list, and then input an estimated XY diameter value of 0.400 micrometers in the box.

Next, click on the green arrow to finish creating the spots, then reselect the spots. Click the Filter icon and select Shortest Distance to Surfaces Surfaces Surfaces X where surfaces X is the surface being analyzed from the drop-down list, ensuring that both lower threshold and upper threshold power buttons are green. Next, input a minimum distance of minus 1.0 micrometer in the lower threshold box and a maximum distance of 0.1 micrometers in the upper threshold box, then click on the Duplicate Section to New Spots button, ensuring that the newly created spots is selected in the upper-left panel of the software window.

Afterward, click on the Gear Tools icon, and then click on the Convex Hull plugin only once and wait for the MATLAB window to appear and then disappear resulting in a solid hull in the 3D view. In the upper-left panel, ensure that Convex Hull of Spots X Selection, Shortest Distance where Spots X Selection is the Newly Created Spots is selected and then click on the hull to select it. Next, click on the Graph Statistics icon.

In the Selection tab, ensure Specific Values is selected from the top drop-down list and then choose Volume from the bottom drop-down list, then record the volume of the hull and save the file, proceeding to the next image. Astrocyte territory volume was measured in astrocytes expressing a membrane-targeted red fluorescent protein. Knockdown of the astrocyte-enriched cell adhesion molecule, hepaCAM, significantly reduces astrocyte territory volume.

Mosaic analysis with double markers was used to generate mice where wild type astrocytes express a red fluorescent protein and hepaCam knockout astrocytes express a green fluorescent protein. The percentage of territory overlap between neighboring red and green fluorescent protein astrocyte pairs was calculated. The mice with genetic modification of hepaCAM and green astrocytes showed a significantly increased percentage of territory overlap volume with their red wild-type neighbors compared to wild-type-only astrocyte pairs.

This result demonstrates that hepaCAM is required for normal astrocyte territory volume, and astrocyte tiling. It is critical to ensure that the cell meets inclusion criteria. An incomplete cell will have a smaller territory volume and may affect your overall results and conclusions.