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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Neuroscience

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

Published: April 20th, 2022

DOI:

10.3791/63804

1Neuroscience Center, University of North Carolina, Chapel Hill, 2Department of Cell Biology and Physiology, 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.

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.

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 channels

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All mice were used in accordance with the Institutional Animal Care and Use Committee (IACUC) at the University of North Carolina at Chapel Hill and the Division of Comparative Medicine (IACUC protocol number 21-116.0). Mice of both sexes at postnatal day 21 (P21) were used for these experiments. CD1 mice were obtained commercially (Table of Materials), and MADM9 WT:WT and MADM9 WT:KO mice were described previously9.

NOTE: This protocol requires brains .......

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Figure 1 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 pr.......

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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 mol.......

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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.

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NameCompanyCatalog NumberComments
#5 forcepsRobozRS-5045
1 mL TB SyringeBecton Dickinson (BD)309623
10x TBS (tris-buffered saline)30 g Tris, 80 g NaCl, 2 g KCl, HCl to pH 7.4, dH2O to 1 L; store at room temperature (RT)
12-well plateGenesee Scientific25-106MP
1x TBS100 mL 10x TBS + 900 mL dH2O; store at RT
1x TBS + Heparin28.2 mg Heparin + 250 mL 1x TBS; store at 4 °C
24-well plateGenesee Scientific25-107MP
30% Sucrose in TBS15 g sucrose, 1x TBS to 50 mL; store at 4 °C
4% PFA (paraformaldehyde) in TBS40 g PFA, 4-6 NaOH pellets, 100 mL 10x TBS, dH2O to 1 L; store at 4 °C
Avertin0.3125 g tri-bromoethanol, 0.625 mL methylbutanol, dH2O to 25 mL; store at 4 °C; discard 2 weeks after making
Blocking and antibody buffer10% goat serum in TBST; store at 4 °C
CD1 miceCharles River022
Collection vial for brainsFisher Scientific03-337-20
Confocal acquisition softwareOlympousFV31S-SW
Confocal microscopeOlympusFV3000RS
CoverslipsFisher Scientific12544E
CryostatThermo ScientificCryoStar NX50
Cryostat bladeThermo Scientific3052835
DAPIInvitrogenD1306
Embedding moldPolysciences18646A-1
Freezing Medium2:1 30% sucrose:OCT; store at RT
GFP antibodyAves LabsGFP1010
GlycerolThermo Scientific158920010
Goat anti-chicken 488InvitrogenA-11039
Goat anti-rabbit 594InvitrogenA11037
Goat SerumGibco16210064
HeparinSigma-AldrichH3149
Hydrochloric acidSigma-Aldrich258148
ImarisBitplaneN/AVersion 9.8.0
MATLABMathWorksN/A
Metal lunch tinAQUARIUSN/AFrom Amazon, "DIY Large Fun Box"
MethylbutanolSigma-Aldrich152463
Micro Dissecting ScissorsRobozRS-5921
Mouting medium20mM Tris pH8.0, 90% Glycerol, 0.5% N-propyl gallate ; store at 4 °C; good for up to 2 months
NailpolishVWR100491-940
N-propyl gallateSigma-Aldrich02370-100G
O.C.T.Fisher Scientific23-730-571
OilOlympusIMMOIL-F30CCSpecific to microscope/objective
Operating Scissors 6"RobozRS-6820
Orbital platform shakerFisher Scientific88861043Minimum speed needed: 25 rpm
PaintbrushBogrinuoN/AFrom Amazon, "Detail Paint Brushes - Miniature Brushes"
ParaformaldehydeSigma-AldrichP6148
Pasteur pipet (5.75")VWR14672-608
Pasteur pipet (9")VWR14672-380
Potassium chlorideSigma-AldrichP9541-500G
Razor bladeFisher Scientific12-640
RFP antibodyRockland600-401-379
Sectioning medium1:1 glycerol:1x TBS; store at RT
SlidesVWR48311-703
Sodium chrloideFisher ScientificBP358-212
Sodium hydroxideSigma-AldrichS5881
SucroseSigma-AldrichS0389
TBST (TBS + Triton X-100)0.2% Triton in 1x TBS; store at RT
Transfer PipetVWR414004-002
Tri-bromoethanolSigma-AldrichT48402
Tris(hydroxymethyl)aminomethaneThermo Scientific424570025
Triton X-100Sigma-Aldrich93443
Triton X-100 (high-quality)Fisher Scientific50-489-120
XTSpotsConvexHullN/AN/Acustom XTension provide as supplementary material
Buffers and Solutions
10x TBSxx mM Tris, xx mM NaCl, xx mM KCl, pH 7.4
1x TBS
1x TBS + Heparinadd xx mg Heparin to xx mL of 1x TBS
4% PFA
30% Sucrose in TBS
Freezing Medium
Sectioning medium
TBST0.2% Triton in 1x TBS
Blocking and antibody buffer10% goat serum in 1x TBST
Mouting medium

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