Microscopy was performed at the UNC Neuroscience Microscopy Core (RRID:SCR_019060), supported in part by funding from the NIH-NINDS Neuroscience Center Support Grant P30 NS045892 and the NIH-NICHD Intellectual and Developmental Disabilities Research Center Support Grant U54 HD079124. Figure 1 was created with BioRender.com. The images and data in Figure 4 are reprinted from a previous publication9 with permission from the publisher.

<ol><li>Stogsdill, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Astrocytic+neuroligins+control+astrocyte+morphogenesis+and+synaptogenesis%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Astrocytic neuroligins control astrocyte morphogenesis and synaptogenesis.</a> Nature. 551 (7679), 192-197 (2017).</li>
<li>Akdemir, E. S., Huang, A. Y., Deneen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Astrocytogenesis%3A+where%2C+when%2C+and+how%5BTitle%5D)+AND+%22F1000Research%22%5BJournal%5D)">Astrocytogenesis: where, when, and how.</a> F1000Research. 9, F1000 Faculty Rev-233 (2020).</li>
<li>Bushong, E. A., Martone, M. E., Jones, Y. Z., Ellisman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protoplasmic+astrocytes+in+CA1+stratum+radiatum+occupy+separate+anatomical+domains%5BTitle%5D)+AND+%22The+Journal+of+Neuroscience%3A+The+Official+Journal+of+the+Society+for+Neuroscience%22%5BJournal%5D)">Protoplasmic astrocytes in CA1 stratum radiatum occupy separate anatomical domains.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 22 (1), 183-192 (2002).</li>
<li>Houades, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Shapes+of+astrocyte+networks+in+the+juvenile+brain%5BTitle%5D)+AND+%22Neuron+Glia+Biology%22%5BJournal%5D)">Shapes of astrocyte networks in the juvenile brain.</a> Neuron Glia Biology. 2 (1), 3-14 (2006).</li>
<li>Zuchero, J. B., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glia+in+mammalian+development+and+disease%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Glia in mammalian development and disease.</a> Development. 142 (22), 3805-3809 (2015).</li>
<li>Srinivasan, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((New+transgenic+mouse+lines+for+selectively+targeting+astrocytes+and+studying+calcium+signals+in+astrocyte+processes+in+situ+and+in+vivo%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">New transgenic mouse lines for selectively targeting astrocytes and studying calcium signals in astrocyte processes in situ and in vivo.</a> Neuron. 92 (6), 1181-1195 (2016).</li>
<li>Testen, A., Kim, R., Reissner, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-resolution+three-dimensional+imaging+of+individual+astrocytes+using+confocal+microscopy%5BTitle%5D)+AND+%22Current+Protocols+in+Neuroscience%22%5BJournal%5D)">High-resolution three-dimensional imaging of individual astrocytes using confocal microscopy.</a> Current Protocols in Neuroscience. 91 (1), 92(2020).</li>
<li>Takano, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemico-genetic+discovery+of+astrocytic+control+of+inhibition+in+vivo%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Chemico-genetic discovery of astrocytic control of inhibition in vivo.</a> Nature. 588 (7837), 296-302 (2020).</li>
<li>Baldwin, K. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HepaCAM+controls+astrocyte+self-organization+and+coupling%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">HepaCAM controls astrocyte self-organization and coupling.</a> Neuron. 109 (15), 2427-2442 (2021).</li>
<li>Amberg, N., Hippenmeyer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+mosaic+dissection+of+candidate+genes+in+mice+using+mosaic+analysis+with+double+markers%5BTitle%5D)+AND+%22STAR+Protocols%22%5BJournal%5D)">Genetic mosaic dissection of candidate genes in mice using mosaic analysis with double markers.</a> STAR Protocols. 2 (4), 100939(2021).</li>
<li>Dumas, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+utero+electroporation+of+multiaddressable+genome-integrating+color+%28MAGIC%29+markers+to+individualize+cortical+mouse+astrocytes%5BTitle%5D)+AND+%22Journal+of+visualized+experiments%3A+JoVE%22%5BJournal%5D)">In utero electroporation of multiaddressable genome-integrating color (MAGIC) markers to individualize cortical mouse astrocytes.</a> Journal of visualized experiments: JoVE. (159), e61110(2020).</li>
<li>Garcia-Marques, J., Nunez-Llaves, R., Lopez-Mascaraque, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NG2-glia+from+pallial+progenitors+produce+the+largest+clonal+clusters+of+the+brain%3A+time+frame+of+clonal+generation+in+cortex+and+olfactory+bulb%5BTitle%5D)+AND+%22The+Journal+of+Neuroscience%3A+The+Official+Journal+of+the+Society+for+Neuroscience%22%5BJournal%5D)">NG2-glia from pallial progenitors produce the largest clonal clusters of the brain: time frame of clonal generation in cortex and olfactory bulb.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 34 (6), 2305-2313 (2014).</li>
<li>Clavreul, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cortical+astrocytes+develop+in+a+plastic+manner+at+both+clonal+and+cellular+levels%5BTitle%5D)+AND+%22Nature+Communication%22%5BJournal%5D)">Cortical astrocytes develop in a plastic manner at both clonal and cellular levels.</a> Nature Communication. 10 (1), 4884(2019).</li>
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</ol>

The authors have no conflicts of interest.

This protocol describes an established method for analyzing astrocyte territory volume and astrocyte tiling in the mouse cortex, detailing all of the major steps beginning with perfusion and ending with image analysis. This protocol requires brains from mice that express fluorescent proteins in a sparse or mosaic population of astrocytes. Outside of this requirement, mice of any age may be used for this protocol, with only minor adjustments to perfusion settings and the volume of freezing media added to the embedding mold. While other methods have been published for analysis of astrocyte branching complexity and volume in brain tissue sections7,11, this protocol has several unique advantages. First, this protocol describes a custom code that can be used to obtain astrocyte territory volume. Astrocyte territory volume is a distinct measurement from astrocyte cell volume, in that it measures the entire space occupied by an astrocyte, regardless of its branching complexity. Furthermore, this protocol describes how to apply the astrocyte territory volume measurement to measure astrocyte territory overlap volume, and therefore astrocyte tiling behavior. Lastly, this protocol provides a detailed discussion of the potential variables that may influence astrocyte territory volume and data collection, including mounting conditions, sample storage conditions, and inclusion and exclusion criteria for imaging and analysis. In addition to these unique features, many aspects of this protocol, including the perfusion, cryosectioning, and immunostaining of free-floating sections, can be broadly applied to staining brain tissue sections of different thicknesses and with different antibody combinations. Below, critical steps, important considerations, troubleshooting, and limitations are discussed in detail.
Sample collection considerations 
One very important consideration for experiments designed to investigate astrocyte morphology is maintaining a consistent time of day for sample collection. Growing evidence indicates that astrocyte functional states are linked to circadian rhythm and sleep behaviors14. To remove any variability that may be introduced by samples being collected at different times of day, perfusions should be planned so that all samples for an experiment are collected at roughly the same time of day, within a time range of 2-3 h. For consistent perfusions, the use of a peristaltic pump is highly recommended. The addition of heparin to the TBS is helpful to reduce the formation of blood clots. While this protocol has not been tested with shorter post-fixation times, post-fixation overnight may not be required, and a shorter post-fixation of 4 h may be considered.
Troubleshooting tips for cryosectioning 
Freezing brains in the 2:1 Sucrose:OCT freezing medium reduces the amount of OCT in the collection wells after sectioning. When frozen, the freezing medium behaves similarly to OCT itself but is less brittle. When freezing the tissue block to the chuck, be sure to press it flat on the chuck immediately after adding OCT to the chuck. If the tissue block breaks off from the chuck during sectioning, use a razor blade to cut a new flat surface on the bottom and refreeze to the chuck. Giving the chuck sufficient time to cool to the cryostat chamber temperature before adding the OCT and making sure that the chuck is clean and dry will improve adherence. When moving tissue sections with the paintbrush, touch the paintbrush to the corner edge of the section, so that is it touching the OCT and not the tissue itself. With a little bit of moisture, the section will stick to the brush. Gently place the tissue into the sectioning medium by touching the opposite edge of the section to the media and allowing it to be pulled into the medium. If the brush touches the freezing medium, wipe it off on a paper towel within the cryostat. A brush that is too wet will make it difficult to transfer sections to the dish. OCT should dissolve once the tissue section is placed in the sectioning medium. If it doesn&#39;t, the cryostat chamber temperature may be too low.
Important considerations for staining and mounting tissue sections 
To improve staining quality, prepare TBST fresh before each experiment. Use a high-quality 10% aqueous Triton source (Table of Materials). Choose a serum that matches the species in which the secondary antibody was made (e.g., use goat serum for a goat anti-rabbit secondary). Antibody solutions are centrifuged prior to use, to pellet and remove any precipitated antibody. Take care not to disturb the pellet when transferring antibody solution from the tube to the plate (this may mean pipetting slightly less than 1 mL of antibody solution per well as to not touch the bottom of the tube with the pipette tip). This step helps to reduce background staining. When sections are placed in TBS:dH2O, most will unfold/untangle themselves, but some may remain folded or twisted. When transferring sections to the slide using a brush, use a P200 tip in the other hand to help guide the tissue section to the appropriate spot. Use the brush to gently unfold or untwist sections so that they lay flat. Sections tend to move toward the aspirating pipet as the liquid is removed. Use the P200 tip to keep mobile sections in place while aspirating. Mounting media must be added to the sections immediately after all the excess liquid is removed and before the sections have time to dry. Allowing the sections to dry even for a few minutes will drastically impact the volume of the cells within the section. After adding the mounting media to the slide, use a pipet to remove any visible bubbles. After adding the coverslip, make sure to remove excess mounting media from the sides, as this will interfere with the ability of the nail polish to dry. Allow the nail polish to dry at room temperature. Wait for at least 2 h or until the next day to image the sections, to ensure that the mounting media has enough time to completely penetrate the thick sections.
Imaging complete astrocytes 
When imaging astrocytes for territory volume analysis, it is critical that the image captures the entire volume of the astrocyte. Many labeled astrocytes in the tissue section will be incomplete astrocytes. Complete astrocytes will be fully contained within the tissue section. To identify complete astrocytes, begin with a focal point within the middle of the astrocyte. Use the focus knob to move to the top of the astrocyte. When the astrocyte is no longer in focus, other features of the tissue should remain in focus. Repeat this process for the bottom of the astrocyte. Do not image incomplete astrocytes. Because of the thickness of the sections, antibody penetration is not as robust in the middle of the section. Labeling may be stronger around the outer edges of the astrocyte and weaker in the center. This is commonly observed and does not impact territory analysis.
Tips for image analysis 
The most important consideration for the image analysis portion of the protocol is to ensure that the convex hull represents only one astrocyte. Follow the inclusion and exclusion criteria each time. Sometimes two astrocytes will have their cell bodies very close to one another. This can be difficult to identify in the 3D view, but is identifiable using the slice view. If there are other fluorescently labeled astrocytes contacting the astrocyte of interest, this can complicate the surface creation process. If a reasonably clear boundary is visible between the two cells (for example, they connect only at one small branch point), the clip tool can be used to make a cut in the surface, and this part of the surface can be deleted. After creating the surface, rotate the cell in 3D view to check for small bits of surface that may be hiding behind the cell, but are not part of the cell. Delete these before proceeding. If the convex hull protrudes drastically to a point outside of the astrocyte territory, there likely is a piece of non-cell surface that was not removed. If the software routinely crashes during surface creation, the surface detail number can be reduced. If it crashes during spots creation, use a region of interest to create spots only near the surface object.
Limitations of the protocol 
While there are many uses and advantages to this protocol, there are also several limitations. This protocol requires mice that already express fluorescent proteins in a sparse population of astrocytes, but does not describe the methods for introducing the expression of fluorescent proteins in astrocytes. The imaging and analysis pipelines are time-consuming and are not well-suited for high-throughput analysis. Furthermore, they are geared specifically toward measuring astrocyte territory volume and tiling, rather than defining the detailed morphological features of individual astrocytes. Others may find it useful to combine approaches and strategies of this protocol with recently published protocols that detail methods for measuring astrocyte morphological complexity7,11. Lastly, the commercial software platforms used in this protocol (i.e., Imaris and MATLAB) are expensive and require a license to use. While large institutions often purchase licenses for these software platforms, this could be cost-prohibitive for smaller institutions and individual labs. Advancements in microscopy, open-source image analysis software, and machine learning algorithms may help make this protocol widely accessible to all labs, and may also improve the efficiency of image acquisition and analysis.

Astrocytes are elaborately branched cells that perform many important functions in the brain1. In the mouse cortex, radial glial stem cells give rise to astrocytes during the late embryonic and early postnatal stages2. During the first three postnatal weeks, astrocytes grow in size and complexity, developing thousands of fine branches that directly interact with synapses1. Concurrently, astrocytes interact with neighboring astrocytes to establish discrete, non-overlapping territories to tile the brain3, while maintaining communication via gap junction channels4. Astrocyte morphology and organization are disrupted in many disease states following insult or injury5, indicating the importance of these processes for proper brain function. Analysis of astrocyte morphological properties during normal development, aging, and disease can provide valuable insights into astrocyte biology and physiology. Furthermore, analysis of astrocyte morphology following genetic manipulation is a valuable tool for discerning the cellular and molecular mechanisms that govern the establishment and maintenance of astrocyte morphological complexity.
Analysis of astrocyte morphology in the mouse brain is complicated by both astrocyte branching complexity and astrocyte tiling. Antibody staining using the intermediate filament glial fibrillary acidic protein (GFAP) as an astrocyte-specific marker captures only the major branches, and vastly underestimates astrocyte morphological complexity1. Other cell-specific markers such as glutamate transporter 1 (GLT-1; slc1a2), glutamine synthetase, or S100&#946; do a better job of labeling astrocyte branches6, but introduce a new problem. Astrocyte territories are largely non-overlapping, but a small degree of overlap exists at the peripheral edges. Because of the complexity of branching, when neighboring astrocytes are labeled the same color, it is impossible to distinguish where one astrocyte ends and the other begins. Sparse or mosaic labeling of astrocytes with endogenous fluorescent proteins solves both problems: the fluorescent marker fills the cell to capture all of the branches and allows for imaging of individual astrocytes that can be distinguished from their neighbors. Several different strategies have been used to achieve sparse fluorescent labeling of astrocytes, with or without genetic manipulation, including viral injection, plasmid electroporation, or transgenic mouse lines. Details on the execution of these strategies are described in previously published studies and protocols1,7,8,9,10,11,12,13.
This article describes a method for measuring astrocyte territory volume from mouse brains with fluorescent labeling in a sparse population of astrocytes (Figure 1). Because the average diameter of an astrocyte in the mouse cortex is approximately 60 &#181;m, 100 &#181;m thick sections are used to improve the efficiency in capturing individual astrocytes in their entirety. Immunostaining is not required but is recommended to enhance the endogenous fluorescent signal for confocal imaging and analysis. Immunostaining may also enable better detection of fine astrocyte branches and reduce photobleaching of endogenous proteins during image acquisition. To improve antibody penetration into the thick sections, and to preserve tissue volume from sectioning through imaging, free-floating tissue sections are used. Analysis of astrocyte territory volume is performed using commercially available image analysis software. Additionally, this protocol describes a method for analysis of astrocyte tiling in tissue sections with mosaic labeling, where neighboring astrocytes express different fluorescent labels. This protocol has been used successfully in several recent studies1,8,9 to characterize astrocyte growth during normal brain development, as well as the impact of genetic manipulation on astrocyte development.

<table><tbody><tr><td>#5 forceps</td><td>Roboz</td><td>RS-5045</td><td></td></tr><tr><td>1 mL TB Syringe</td><td>Becton Dickinson (BD)</td><td>309623</td><td></td></tr><tr><td>10x TBS (tris-buffered saline)</td><td></td><td></td><td>30 g Tris, 80 g NaCl, 2 g KCl, HCl to pH 7.4, dH&lt;sub&gt;2&lt;/sub&gt;O to 1 L; store at room temperature (RT)</td></tr><tr><td>12-well plate</td><td>Genesee Scientific</td><td>25-106MP</td><td></td></tr><tr><td>1x TBS</td><td></td><td></td><td>100 mL 10x TBS + 900 mL dH&lt;sub&gt;2&lt;/sub&gt;O; store at RT</td></tr><tr><td>1x TBS + Heparin</td><td></td><td></td><td>28.2 mg Heparin + 250 mL 1x TBS; store at 4 &amp;deg;C</td></tr><tr><td>24-well plate</td><td>Genesee Scientific</td><td>25-107MP</td><td></td></tr><tr><td>30% Sucrose in TBS</td><td></td><td></td><td>15 g sucrose, 1x TBS to 50 mL; store at 4 &amp;deg;C</td></tr><tr><td>4% PFA (paraformaldehyde) in TBS</td><td></td><td></td><td>40 g PFA, 4-6 NaOH pellets, 100 mL 10x TBS, dH&lt;sub&gt;2&lt;/sub&gt;O to 1 L; store at 4 &amp;deg;C</td></tr><tr><td>Avertin</td><td></td><td></td><td>0.3125 g tri-bromoethanol, 0.625 mL methylbutanol, dH&lt;sub&gt;2&lt;/sub&gt;O to 25 mL; store at 4 &amp;deg;C; discard 2 weeks after making</td></tr><tr><td>Blocking and antibody buffer</td><td></td><td></td><td>10% goat serum in TBST; store at 4 &amp;deg;C</td></tr><tr><td>CD1 mice</td><td>Charles River</td><td>022</td><td></td></tr><tr><td>Collection vial for brains</td><td>Fisher Scientific</td><td>03-337-20</td><td></td></tr><tr><td>Confocal acquisition software</td><td>Olympus</td><td>FV31S-SW</td><td></td></tr><tr><td>Confocal microscope</td><td>Olympus</td><td>FV3000RS</td><td></td></tr><tr><td>Coverslips</td><td>Fisher Scientific</td><td>12544E</td><td></td></tr><tr><td>Cryostat</td><td>Thermo Scientific</td><td>CryoStar NX50</td><td></td></tr><tr><td>Cryostat blade</td><td>Thermo Scientific</td><td>3052835</td><td></td></tr><tr><td>DAPI</td><td>Invitrogen</td><td>D1306</td><td></td></tr><tr><td>Embedding mold</td><td>Polysciences</td><td>18646A-1</td><td></td></tr><tr><td>Freezing Medium</td><td></td><td></td><td>2:1 30% sucrose:OCT; store at RT</td></tr><tr><td>GFP antibody</td><td>Aves Labs</td><td>GFP1010</td><td></td></tr><tr><td>Glycerol</td><td>Thermo Scientific</td><td>158920010</td><td></td></tr><tr><td>Goat anti-chicken 488</td><td>Invitrogen</td><td>A-11039</td><td></td></tr><tr><td>Goat anti-rabbit 594</td><td>Invitrogen</td><td>A11037</td><td></td></tr><tr><td>Goat Serum</td><td>Gibco</td><td>16210064</td><td></td></tr><tr><td>Heparin</td><td>Sigma-Aldrich</td><td>H3149</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Sigma-Aldrich</td><td>258148</td><td></td></tr><tr><td>Imaris</td><td>Bitplane</td><td>N/A</td><td>Version 9.8.0</td></tr><tr><td>MATLAB</td><td>MathWorks</td><td>N/A</td><td></td></tr><tr><td>Metal lunch tin</td><td>AQUARIUS</td><td>N/A</td><td>From Amazon, &quot;DIY Large Fun Box&quot;</td></tr><tr><td>Methylbutanol</td><td>Sigma-Aldrich</td><td>152463</td><td></td></tr><tr><td>Micro Dissecting Scissors</td><td>Roboz</td><td>RS-5921</td><td></td></tr><tr><td>Mounting medium</td><td></td><td></td><td>20 mM Tris pH 8.0, 90% Glycerol, 0.5% N-propyl gallate; store at 4 &amp;deg;C; good for up to 2 months</td></tr><tr><td>Nail polish</td><td>VWR</td><td>100491-940</td><td></td></tr><tr><td>N-propyl gallate</td><td>Sigma-Aldrich</td><td>02370-100G</td><td></td></tr><tr><td>O.C.T.</td><td>Fisher Scientific</td><td>23-730-571</td><td></td></tr><tr><td>Oil</td><td>Olympus</td><td>IMMOIL-F30CC</td><td>Specific to microscope/objective</td></tr><tr><td>Operating Scissors 6&quot;</td><td>Roboz</td><td>RS-6820</td><td></td></tr><tr><td>Orbital platform shaker</td><td>Fisher Scientific</td><td>88861043</td><td>Minimum speed needed: 25 rpm</td></tr><tr><td>Paintbrush</td><td>Bogrinuo</td><td>N/A</td><td>From Amazon, &quot;Detail Paint Brushes - Miniature Brushes&quot;</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td></td></tr><tr><td>Pasteur pipet (5.75&quot;)</td><td>VWR</td><td>14672-608</td><td></td></tr><tr><td>Pasteur pipet (9&quot;)</td><td>VWR</td><td>14672-380</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich</td><td>P9541-500G</td><td></td></tr><tr><td>Razor blade</td><td>Fisher Scientific</td><td>12-640</td><td></td></tr><tr><td>RFP antibody</td><td>Rockland</td><td>600-401-379</td><td></td></tr><tr><td>Sectioning medium</td><td></td><td></td><td>1:1 glycerol:1x TBS; store at RT</td></tr><tr><td>Slides</td><td>VWR</td><td>48311-703</td><td></td></tr><tr><td>Sodium chrloide</td><td>Fisher Scientific</td><td>BP358-212</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>S5881</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S0389</td><td></td></tr><tr><td>TBST (TBS + Triton X-100)</td><td></td><td></td><td>0.2% Triton in 1x TBS; store at RT</td></tr><tr><td>Transfer Pipet</td><td>VWR</td><td>414004-002</td><td></td></tr><tr><td>Tri-bromoethanol</td><td>Sigma-Aldrich</td><td>T48402</td><td></td></tr><tr><td>Tris(hydroxymethyl)aminomethane</td><td>Thermo Scientific</td><td>424570025</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>93443</td><td></td></tr><tr><td>Triton X-100 (high-quality)</td><td>Fisher Scientific</td><td>50-489-120</td><td></td></tr><tr><td>XTSpotsConvexHull</td><td>N/A</td><td>N/A</td><td>custom XTension provide as supplementary material</td></tr></tbody></table>

analysis of astrocyte territory volume and tiling in thick free-floating tissue sections

Astrocytes possess an astounding degree of morphological complexity that enables them to interact with nearly every type of cell and structure within the brain. Through these interactions, astrocytes actively regulate many critical brain functions, including synapse formation, neurotransmission, and ion homeostasis. In the rodent brain, astrocytes grow in size and complexity during the first three postnatal weeks and establish distinct, non-overlapping territories to tile the brain. This protocol provides an established method for analyzing astrocyte territory volume and astrocyte tiling using free-floating tissue sections from the mouse brain. First, this protocol describes the steps for tissue collection, cryosectioning, and immunostaining of free-floating tissue sections. Second, this protocol describes image acquisition and analysis of astrocyte territory volume and territory overlap volume, using commercially available image analysis software. Lastly, this manuscript discusses the advantages, important considerations, common pitfalls, and limitations of these methods. This protocol requires brain tissue with sparse or mosaic fluorescent labeling of astrocytes, and is designed to be used with common lab equipment, confocal microscopy, and commercially available image analysis software.

Figure 1&#160;presents a schematic outline of the major steps and workflow for this protocol. Figure 2 shows screenshots of key steps using the image analysis software to generate a surface, generate spots close to the surface, and generate a convex hull. Figure 3 demonstrates the application of this technique to determine astrocyte territory overlap/tiling. In Figure 4, representative results from a previously published manuscript9 demonstrate the application of this protocol. In Figure 4A and Figure 4B, knockdown of the astrocyte-enriched cell adhesion molecule hepaCAM significantly reduces astrocyte territory volume. In Figure 4C and Figure 4D, Mosaic Analysis with Double Markers (MADM) was used to introduce sparse mosaic labeling and concurrent genetic modification into the mouse cortex. With MADM, mice were generated where wild-type astrocytes express a red fluorescent protein (RFP) and Hepacam knockout astrocytes express a green fluorescent protein (GFP). Using the protocol described above, the percentage of territory overlap between neighboring RFP and GFP astrocyte pairs (WT:KO) was calculated. As a control, this was compared to neighboring RFP and GFP astrocyte pairs in mice with no genetic modification of Hepacam (WT:WT). WT:KO astrocyte pairs showed a significantly increased percentage of territory overlap volume compared to WT:WT astrocyte pairs. Collectively, these results demonstrate that hepaCAM is required for normal astrocyte territory volume and astrocyte tiling.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63804/63804fig01.jpg" /> 
Figure 1: Overview of workflow for analyzing astrocyte territory volume. (A) The protocol requires a mouse with sparse or mosaic fluorescent labeling of astrocytes. (B) The mouse is perfused and then the brain is resected, processed, and frozen. (C) The frozen brain is sectioned using a cryostat and (D) the free-floating tissue sections are collected. (E) After sectioning, the floating tissue sections are stained by immunohistochemistry and then (F) mounted onto slides. (G) Astrocytes within the mounted tissue sections are imaged using confocal microscopy. (H) Finally, the imaged cells&#39; territory volumes are quantified using image analysis software. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63804/63804fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63804/63804fig02.jpg" /> 
Figure 2: Key steps in image analysis procedure for astrocyte territory volume. (A) View of cell with region of interest box that excludes other signal in the image from the analysis procedure. (B) An acceptable relative threshold for surface creation (gray) using signal (red). (C) Completed surface with main cell selected (yellow). Other parts of the surface that aren&#39;t the cell will be deleted (red). (D) Translucent surface with spots created. (E) Close-up of the translucent surface with spots created to show an acceptable distribution of spots relative to the surface. (F) Final convex hull. Scale bars = 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63804/63804fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63804/63804fig03.jpg" /> 
Figure 3: Key steps in astrocyte territory overlap analysis. (A) Representative image of two astrocytes expressing different fluorescent markers, GFP (green) and RFP (magenta). (B) Generation of a convex hull for each astrocyte. (C) View of masked GFP and RFP channels, with original channels removed. (D) Example of thresholding using the &#34;Coloc&#34; tool to create a &#34;Colocalization Result&#34; channel. The &#34;colocalized&#34; signal is shown in gray. (E) View of the two masked channels. (F) A rotated view of the convex hull of the &#34;Colocalization Result&#34; channel (gray) which represents the territory overlap volume. Scale bars = 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63804/63804fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63804/63804fig04.jpg" /> 
Figure 4: Successful application of the protocol to measure astrocyte territory volume and astrocyte territory overlap. (A) Astrocytes in the visual cortex of wild-type CD1 mice at postnatal day 21, expressing mCherry-CAAX (cyan) and a control shRNA (shScramble) or an shRNA targeting hepaCAM (shHepacam). The astrocyte territory is outlined in red. (B) Loss of hepaCAM significantly reduces astrocyte territory volume. Data points represent individual mouse averages. Error bars are &#177; s.e.m. Nested t-test. (C) Neighboring astrocytes in the mouse cortex at postnatal day 21 expressing GFP (green) or RFP (magenta). In MADM9 WT:WT mice, both astrocytes are wild-type. In MADM9 WT:KO mice, the green astrocyte is wild-type and the magenta astrocyte is null for Hepacam. Territory overlap volume is shown in blue. (MADM9 WT:WT genotype: MADM9TG/GT; EMX-CreTg/0; Hepacam+/+. MADM9 WT:KO genotype: MADM9TG/GT; EMX-CreTg/0; Hepacam+/-). (D) Quantification of the percentage of territory volume overlap. Data points represent individual mouse averages. Error bars are &#177; s.e.m. Nested t-test. Scale bars = 20 &#181;m. The panels in this figure are reprinted from Baldwin et al9 with permission from the publisher. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63804/63804fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental File. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63804/XTSpotsConvexHull.m" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Analysis of Astrocyte Territory Volume and Tiling in Thick Free-Floating Tissue Sections at JoVE.com

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.

Astrocytes possess an astounding degree of morphological complexity that enables them to interact with nearly every type of cell and structure within the ...

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3.4K Views.  University of North Carolina, Chapel Hill. 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.

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

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Biology

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify their functional and structural properties, a condition called reactive astrogliosis. Here, a protocol for assessment of the morphological properties of astrocytes is presented. This protocol includes quantification of 12 different parameters i.e. the surface area and volume of the tissue covered by an astrocyte (astrocyte territory), the entire astrocyte including branches, cell body, and nucleus, as well as total length and number of branches, the intensity of fluorescence immunoreactivity of antibodies used for astrocyte detection, and astrocyte density (number/1,000 &#181;m2). For this purpose three-dimensional (3D) confocal microscopic images were created, and 3D image analysis software such as Volocity 6.3 was used for measurements. Rat brain tissue exposed to amyloid beta1-40 (A&#946;1-40) with or without a therapeutic&#160;intervention was used to present the method. This protocol can also be used for 3D morphometric analysis of other cells from either in vivo or in vitro conditions.

Astrocytes in the CNS change their functional and structural properties in response to harmful stimuli. This report presents a protocol for assessment of three-dimensional astrocyte morphology in diseased conditions or after therapeutic interventions.

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

Analyzing the Size, Shape, and Directionality of Networks of Coupled Astrocytes

It has become increasingly clear that astrocytes modulate neuronal function not only at the synaptic and single-cell levels, but also at the network level. Astrocytes are strongly connected to each other through gap junctions and coupling through these junctions is dynamic and highly regulated. An emerging concept is that astrocytic functions are specialized and adapted to the functions of the neuronal circuit with which they are associated. Therefore, methods to measure various parameters of astrocytic networks are needed to better describe the rules governing their communication and coupling and to further understand their functions.
Here, using the image analysis software (e.g., ImageJFIJI), we describe a method to analyze confocal images of astrocytic networks revealed by dye-coupling. These methods allow for 1) an automated and unbiased detection of labeled cells, 2) calculation of the size of the network, 3) computation of the preferential orientation of dye spread within the network, and 4) repositioning of the network within the area of interest.
This analysis can be used to characterize astrocytic networks of a particular area, compare networks of different areas associated to different functions, or compare networks obtained under different conditions that have different effects on coupling. These observations may lead to important functional considerations. For instance, we analyze the astrocytic networks of a trigeminal nucleus, where we have previously shown that astrocytic coupling is essential for the ability of neurons to switch their firing patterns from tonic to rhythmic bursting1. By measuring the size, confinement, and preferential orientation of astrocytic networks in this nucleus, we can build hypotheses about functional domains that they circumscribe. Several studies suggest that several other brain areas, including the barrel cortex, lateral superior olive, olfactory glomeruli, and sensory nuclei in the thalamus and visual cortex, to name a few, may benefit from a similar analysis.

Here we present a protocol to assess the organization of astrocytic networks. The described method minimizes bias to provide descriptive measures of these networks such as cell count, size, area, and position within a nucleus. Anisotropy is assessed with a vectorial analysis.

It has become increasingly clear that astrocytes modulate neuronal function not only at the synaptic and single-cell levels, but also at the network ...

A Method for 3D Reconstruction and Virtual Reality Analysis of Glial and Neuronal Cells

Serial sectioning and subsequent high-resolution imaging of biological tissue using electron microscopy (EM) allow for the segmentation and reconstruction of high-resolution imaged stacks to reveal ultrastructural patterns that could not be resolved using 2D images. Indeed, the latter might lead to a misinterpretation of morphologies, like in the case of mitochondria; the use of 3D models is, therefore, more and more common and applied to the formulation of morphology-based functional hypotheses. To date, the use of 3D models generated from light or electron image stacks makes qualitative, visual assessments, as well as quantification, more convenient to be performed directly in 3D. As these models are often extremely complex, a virtual reality environment is also important to be set up to overcome occlusion and to take full advantage of the 3D structure. Here, a step-by-step guide from image segmentation to reconstruction and analysis is described in detail.

The described pipeline is designed for the segmentation of electron microscopy datasets larger than gigabytes, to extract whole-cell morphologies. Once the cells are reconstructed in 3D, customized software designed around individual needs can be used to perform a qualitative and quantitative analysis directly in 3D, also using virtual reality to overcome view occlusion.

Serial sectioning and subsequent high-resolution imaging of biological tissue using electron microscopy (EM) allow for the segmentation and reconstruction ...

Visualizing Astrocyte Morphology Using Lucifer Yellow Iontophoresis

Astrocytes are essential components of neural circuits. They tile the entire central nervous system (CNS) and are involved in a variety of functions, which include neurotransmitter clearance, ion regulation, synaptic modulation, metabolic support to neurons, and blood flow regulation. Astrocytes are complex cells that have a soma, several major branches, and numerous fine processes that contact diverse cellular elements within the neuropil. In order to assess the morphology of astrocytes, it is necessary to have a reliable and reproducible method to visualize their structure. We report a reliable&#160;protocol to perform intracellular iontophoresis of astrocytes using fluorescent Lucifer yellow (LY) dye in lightly fixed brain tissue from adult mice. This method has several features that are useful to characterize astrocyte morphology. It allows for three-dimensional reconstruction of individual astrocytes, which is useful to perform morphological analyses on different aspects of their structure. Immunohistochemistry together with LY iontophoresis can also be utilized to understand the interaction of astrocytes with different components of nervous system and to evaluate the expression of proteins within the labelled astrocytes. This protocol can be implemented in a variety of mouse models of CNS disorders to rigorously examine astrocyte morphology with light microscopy. LY iontophoresis provides an experimental approach to evaluate astrocyte structure, especially in the context of injury or disease where these cells are proposed to undergo significant morphological changes.

Astrocytes are morphologically complex cells, exemplified by their multiple processes and bushy territories. To analyze their elaborate morphology, we present a reliable protocol to perform intracellular Lucifer yellow iontophoresis in lightly fixed tissue.

Astrocytes are essential components of neural circuits. They tile the entire central nervous system (CNS) and are involved in a variety of functions, ...

Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in brain homeostasis, astrocytes supply neurons with vital metabolites and buffer extracellular water, ions, and glutamate. An integral component of the &#8220;tri-partite&#8221; synapse, astrocytes are also critical in the formation, pruning, maintenance, and modulation of synapses. To enable these highly interactive functions, astrocytes communicate among themselves and with other glial cells, neurons, the brain vasculature, and the extracellular environment through a multitude of specialized membrane proteins that include cell adhesion molecules, aquaporins, ion channels, neurotransmitter transporters, and gap junction molecules. To support this dynamic flux, astrocytes, like neurons, rely on tightly coordinated and efficient intracellular transport. Unlike neurons,&#160;where intracellular trafficking has been extensively delineated, microtubule-based transport in astrocytes has been less studied. Nonetheless, exo- and endocytic trafficking of cell membrane proteins and intracellular organelle transport orchestrates astrocytes&#8217; normal biology, and these processes are often affected in disease or in response to injury. Here we present a straightforward protocol to culture high quality murine astrocytes, to fluorescently label astrocytic proteins and organelles of interest, and to record their intracellular transport dynamics using time-lapse confocal microscopy. We also demonstrate how to extract and quantify relevant transport parameters from the acquired movies using available image analysis software (i.e., ImageJ/FIJI) plugins.

Here we describe an in vitro live-imaging method to visualize intracellular transport of organelles and trafficking of plasma membrane proteins in murine astrocytes. This protocol also presents an image analysis methodology to determine cargo transport itineraries and kinetics.

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in ...

In Utero Electroporation of Multiaddressable Genome-Integrating Color (MAGIC) Markers to Individualize Cortical Mouse Astrocytes

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at adult stages. Thus far, no immunostaining strategy can single them out and segment their morphology in mature animals and over the course of corticogenesis. Cortical PrA originate from progenitors located in the dorsal pallium and can easily be targeted using in utero electroporation of integrative vectors. A protocol is presented here to label these cells with the multiaddressable genome-integrating color (MAGIC) Markers strategy, which relies on piggyBac/Tol2 transposition and Cre/lox recombination to stochastically express distinct fluorescent proteins (blue, cyan, yellow, and red) addressed to specific subcellular compartments. This multicolor fate mapping strategy enables to mark in situ nearby cortical progenitors with combinations of color markers prior to the start of gliogenesis and to track their descendants, including astrocytes, from embryonic to adult stages at the individual cell level. Semi-sparse labeling achieved by adjusting the concentration of electroporated vectors and color contrasts provided by the Multiaddressable Genome-Integrating Color Markers (MAGIC Markers or MM) enable to individualize astrocytes and single out their territory and complex morphology despite their dense anatomical arrangement. Presented here is a comprehensive experimental workflow including the details of the electroporation procedure, multichannel image stacks acquisition by confocal microscopy, and computer-assisted three-dimensional segmentation that will enable the experimenter to assess individual PrA volume and morphology. In summary, electroporation of MAGIC Markers provides a convenient method to individually label numerous astrocytes and gain access to their anatomical features at different developmental stages. This technique will be useful to analyze cortical astrocyte morphological properties in various mouse models without resorting to complex crosses with transgenic reporter lines.

In Utero Electroporation of Multiaddressable Genome-Integrating Color MAGIC Markers to Individualize Cortical Mouse Astrocytes

Astrocytes tile the cerebral cortex uniformly, making the analysis of their complex morphology challenging at the cellular level. The protocol provided here uses multicolor labeling based on in utero electroporation to single out cortical astrocytes and analyze their volume and morphology with a user-friendly image analysis pipeline.

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at ...

Investigation of Spatial Interaction Between Astrocytes and Neurons in Cleared Brains

Combining viral vector transduction and tissue clearing using the CLARITY method makes it possible to simultaneously investigate several types of brain cells and their interactions. Viral vector transduction enables the marking of diverse cell types in different fluorescence colors within the same tissue. Cells can be identified genetically by activity or projection. Using a modified CLARITY protocol, the potential sample size of astrocytes and neurons has grown by 2-3 orders of magnitude. The use of CLARITY allows the imaging of complete astrocytes, which are too large to fit in their entirety in slices, and the examination of the somata with all their processes. In addition, it provides the opportunity to investigate the spatial interaction between astrocytes and different neuronal cell types, namely, the number of pyramidal neurons in each astrocytic domain or the proximity between astrocytes and specific inhibitory neuron populations. This paper describes, in detail, how these methods are to be applied.

Combining viral vector transduction and brain clearing using the CLARITY method allows the investigation of a large number of neurons and astrocytes simultaneously.

Combining viral vector transduction and tissue clearing using the CLARITY method makes it possible to simultaneously investigate several types of brain ...

Visualizing Astrocyte Morphology Using Fluorescent Dye Iontophoresis in a Fixed Mouse Brain Slice

Source:&#160;<a style='text-decoration:none;' href='https://app-jove-com.remotexs.ntu.edu.sg/v/60225/visualizing-astrocyte-morphology-using-lucifer-yellow-iontophoresis'><span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>Moye, S. L.,&#160;et.al.<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> Visualizing Astrocyte Morphology Using Lucifer Yellow Iontophoresis.&#160;J. Vis. Exp.<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> (2019).</a> &#160;This video demonstrates the use of fluorescent dye iontophoresis to study astrocyte morphology in fixed brain slices. It details the piercing of the astrocyte soma with an electrode to deliver an anionic, hydrophilic dye into the cytoplasm under a low-voltage current, enabling the dye to diffuse and label the soma and branches. Confocal microscopy is then used to visualize the labeled astrocytes, providing morphological insights.

Source:&#160;Moye, S. L.,&#160;et.al. Visualizing Astrocyte Morphology Using Lucifer Yellow Iontophoresis.&#160;J. Vis. Exp. (2019).&#160;This video ...

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Neuroimaging

Advanced Microscopy & Imaging Technologies

Immunostaining of Fluorescently Labeled Astrocytes in a Mouse Brain Tissue Section

Source: Eaker, A. R., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/63804">Analysis of Astrocyte Territory Volume and Tiling in Thick Free-Floating Tissue Sections.</a> J. Vis. Exp. (2022)
This video demonstrates the procedure for immunostaining a fixed mouse brain tissue section with fluorescently labeled astrocytes using primary and secondary antibodies and capture the images of the stained astrocytes through confocal microscopy.

Source: Eaker, A. R., et al. Analysis of Astrocyte Territory Volume and Tiling in Thick Free-Floating Tissue Sections. J. Vis. Exp. (2022)
This video ...

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

Summary

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Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

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A Method for 3D Reconstruction and Virtual Reality Analysis of Glial and Neuronal Cells

Visualizing Astrocyte Morphology Using Lucifer Yellow Iontophoresis

Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes

In Utero Electroporation of Multiaddressable Genome-Integrating Color (MAGIC) Markers to Individualize Cortical Mouse Astrocytes

Investigation of Spatial Interaction Between Astrocytes and Neurons in Cleared Brains

Visualizing Astrocyte Morphology Using Fluorescent Dye Iontophoresis in a Fixed Mouse Brain Slice

Immunostaining of Fluorescently Labeled Astrocytes in a Mouse Brain Tissue Section