Name: Astrositler Üç boyutlu Konfokal Morfometrik Analizi Protokolü (Video) | JoVE Journal
Uploaded: 2015-12-11T14:00:00.000+00:00
Duration: 9 min 13 s
Description: Öğrenin, konfokal mikroskopi ile astrosit morfolojisini üç boyutlu analiz etmeyi. Bu yöntem, patolojik koşullarda veya terapötik müdahaleler sonrası hücresel mimari değişikliklerini değerlendirmeye yardımcı olur.

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The authors have nothing to disclose.

This study was carried out in accordance with the policies set forth in the Guide for the Care and Use of Laboratory Animals (NIH) approved by Ethic Committee of Iran University of Medical Sciences (Tehran, Iran).
1. Animals, Surgery and Specimen Preparations
NOTE: Prepare brain tissue for 3D confocal microscopic analysis.
<ol>
	<li>Divide animals randomly into two groups: A&#946;1-40&#8210;injection (n = 8), and A&#946;1-40&#8210;injection with genistein treatment (n = 8); administer genistein (10 mg/kg diluted in a vehicle such as polyethoxylated castor oil) by gavage 1 hr before surgery.</li>
	<li>Anesthetize the animals with ketamine (100 mg/kg) and xylazine (10 mg/kg). Confirm proper anesthesia by lack of withdrawal reflex after pinching the toe. Use ointment on eyes during the surgery to prevent dryness.</li>
	<li>Place animals in stereotaxic apparatus and shave head. Apply iodine solution to clean the scalp before incision and keep the operation site sterile during the surgery to reduce the risk of infection. Inject A&#946;1-40 stereotaxically (4 &#181;l) in the hippocampus at &#8722;3.5 mm posterior to bregma, &#177; 2 mm lateral to midline, and &#8722;2.8 mm below dura, according to rat brain atlas8. For stereotaxic method please see previous publication9.</li>
	<li>Provide post-operative observation/care until the animals are fully conscious to ensure the comfort of the animals. Keep the animals warm during recovery, and do not return them to a cage with other animals until full recovery. Pay attention to any sign of infection in the animals after surgery.</li>
	<li>Anesthetize the animals deeply by ketamine (150 mg/kg) three weeks after surgery. Perform transcardial fixation by perfusing the animals with 0.9% saline followed by 4% paraformaldehyde in 0.1 M PBS (pH = 7.4), then remove and fix brain as described in10.</li>
	<li>Post-fix the brains in 4% paraformaldehyde for 2-3 days at 4 &#730;C.</li>
	<li>Embed the tissue in paraffin blocks by use of tissue embedding equipment, and avoid under-filling or over-filling the cassette since it may interfere with correct alignment or sectioning. Pay attention to the orientation of the tissue in the paraffin block. 
	NOTE: The rat hippocampus, for example, is close to posterior part of the cerebral hemisphere. Thus, the tissue should be embedded in a way that sectioning starts at posterior part of the brain. Use the Paxinos atlas8 as a guide to reach the desired anatomical structure.</li>
	<li>Place the microtome away from air drafts or doorways since air movements make the handling of sections difficult.</li>
	<li>Use sections with a thickness &#8805;20 &#181;m as this depth with be necessary to produce Z-Stack images of complete astrocytes. Do not use the first couple sections, as they may have undesired thickness due to thermal expansion.</li>
	<li>Place the sections on the surface of warm water (5 &#177; 2 &#730;C, below the melting temperature for paraffin) just long enough to flatten the sections. 
	NOTE: Over expansion of the section can disturb the morphology of the structure. Use tiny paintbrush to carefully transfer sections. Collect two to three sections on each slide.</li>
	<li>Store slides in a rack in an upright position and let them dry at 37 &#730;C for several hr or O/N.</li>
</ol>
2. Immunohistochemistry
<ol>
	<li>Deparaffinize sections in xylene, 2 x 10 min, and rehydrate through alcohol gradient (99.8%, 95%, and 70%, 10 min each) and PBS.</li>
	<li>Incubate with antibodies against glial fibrillary acidic protein (GFAP) diluted 1:1,500 in PBS containing 0.25% bovine serum albumin (BSA), 0.25% Triton X-100 and 3.5% normal serum (O/N at 4 &#730;C).</li>
	<li>Wash sections in PBS for 3 x 5 min.</li>
	<li>Incubate with secondary alkaline phosphate-conjugated antibodies for 1h (1:100, 21 &#730;C, diluted in PBS).</li>
	<li>Wash the sections in PBS for 3 x 5 min. 
	NOTE: It is important to wash the sections properly after secondary antibodies to minimize background staining.</li>
	<li>Incubate sections in darkness with Liquid Permanent Red Chromogen (15-20 min). Note: Prepare the solution no more than 30 min before use.</li>
	<li>Wash sections in PBS for 3 x 5 min.</li>
	<li>Finally, stain the nuclei with 4-6-diamidino-2-phenylindole (DAPI; 1:500, diluted in PBS) prior to cover-slipping. Store the slides in the refrigerator 24-48 hr before microscopy.</li>
</ol>
3. Confocal Microscopy
NOTE: For quantitative evaluation (see below), select an astrocyte with a clearly visible DAPI-stained nucleus with a minimum of overlapping branches to reduce the risk of errors. Producing Z-Stack images is time consuming. Be patient and do not stop during processing. Upright confocal laser scanning microscope is used to create 3D images from astrocytes.
<ol>
	<li>Put the slide on the microscope-stage, and select the 63X objective.</li>
	<li>Open the imaging software and click on Start system. Click on the Locate tab. Go to Assign, and select GFP to adjust the proper light.</li>
	<li>Open the Shutter to reflect light. Find the Reflector Revolver on the right side of Shutter and choose the color that should be reflected by astrocytes (green, red or violet); red color (FSet20 wf) was used here. 
	NOTE: Do not forget to focus the picture directly in the ocular of the microscope.</li>
	<li>To find the best focus, click on All closed, put the microscope pin in screen mode, and click on the Acquisition tab.</li>
	<li>Click on Smart Setup under the Acquisition tab; a window opens. Select the proper fluorophore (i.e. DAPI and Alexa Fluor 555, in the current project) from the list. The color is automatically selected.</li>
	<li>Click on Best signal, and Apply, then click on Set Exposure; the computer will then automatically set the exposure parameters.</li>
	<li>For optimizing the image, go to the Channels window. Unmark Track2, highlight Track1 and click on Live. Focus the image if needed.</li>
	<li>In the Channels window adjust the following keys; click on&#160;1AU to automatically optimize the pinhole. It is very important to do this step otherwise the confocal images will be non-optimal. Adjust Gain to have the best intensity (keep this setting below 800 to reduce extra noise). Finally, set the Digital Gain between 2 and 3.</li>
	<li>Unmark Track1, click on Track2 and repeat Step 3.8 for Track2.&#160;Then stop Live.</li>
	<li>Go to the Acquisition Mode window. Improve the image by optimizing Frame Size and Averaging. In order to change frame size go to X*Y and select 1,024 x 1,024. Choose a slow Speed to create a better image.</li>
	<li>Go to Averaging and select a Number &#8805;4. This averaging value indicates the number of scans that will be averaged to produce the acquired image. Now click on the Snap button, and save the captured 2D image.</li>
	<li>For 3D imaging, mark the Z-Stack item under Smart Setup causing the Z-Stack window to open.</li>
	<li>Set the first and last positions for the Z-Stack, i.e. the depth of the cell. First click on Live. Then use the focus drive of the microscope to focus on the uppermost position of astrocyte in the tissue, where the Z-Stack is to start. Click on Set First. Then focus down to the lowest position of the astrocyte in the tissue, where Z-Stack scanning should be stopped. Click on Set Last. Then stop Live.</li>
	<li>Choose the Interval; 1.01 &#181;m is used in the current study; choosing interval can be done by clicking on Optimal to set the number of slices.</li>
	<li>Click on Start Experiment. Save the images once the scan is complete. This image will be used for the measurement of the following parameters: surface area and volume of astrocyte territory, the entire astrocyte including branches, cell body, and nucleus.</li>
	<li>Acquire a 2D image using a 10X or 20X objective as described in Steps 3.6-3.11. This image can be used for the measurement the intensity of fluorescence immunoreactivity of antibodies, and astrocyte density (number/1,000 &#181;m2).</li>
</ol>
4. Astrocyte Density and GFAP+ Fluorescence Intensity
<ol>
	<li>Open the 20X 2D image. Click on Histo (Hisogram) on the left side of image window.</li>
	<li>To calculate astrocyte density, select three consecutive visual fields under 20X objective. Count the number of astrocytes that exhibit a clear soma with minimum, overlapping branches in the fields.</li>
	<li>To quantify fluorescence intensity, select a proper tool (rectangle or circle) on the footer of image window, and choose an area for the measurement. Note the mean value and standard deviation appear automatically under the image. In the current study, a 0.7 mm2 rectangular area between striatum and medial blade of dentate gyrus was used.</li>
</ol>
5. Astrocyte Branches
<ol>
	<li>To quantify the length and total number of branches, use 3D images (acquired with 20X) in the image analyzing software. Draw a line manually along the length of each branch of the selected astrocyte. Then select the measurement-tool that is specialized for measuring line-length. In order to quantify the number of branches, select the count-tool to count the number of marked items.</li>
</ol>
6. Volume and Surface Area
NOTE: Marking a structure manually in 3D image analysis software requires focus and training. Practice several times before starting an experiment.
<ol>
	<li>To quantify the volume and surface area of the astrocyte nucleus, soma, cell body and territory, transfer the 3D images (acquired in Steps 3.1-3.16) to a 3D image analysis software such as Volocity 6.1.</li>
	<li>Open the software and create a new library. Drag all images to the left gray column in the new library. Then, select one 3D image. On the right, there are different channels with different colors. Turn on one channel of interest; for example the blue channel for DAPI when measuring the nucleus. Change the brightness manually to get an optimal contrast.</li>
	<li>Select the Tools menu, go to Make Volume and produce a single 3D image out of Z-stack images. Click on Properties in the Edit menu and be sure that X, Y and Z properties are presented; otherwise it is impossible to carry out 3D analysis.</li>
	<li>Click on Freehand RIO tool icon in the toolbar. To measure the volume and surface area of astrocyte nucleus draw a line around the DAPI&#8210;labeled nucleus.</li>
	<li>To measure the volume and surface area of astrocyte cell body draw a line around the soma excluding branches.</li>
	<li>To measure the volume and surface area of entire astrocyte (cell body including branches) draw a line around the soma following the surface of all branches.</li>
	<li>To assess the volume and surface area of astrocyte territory draw a line between tips of the branches. 
	NOTE: Press Delete on the keyboard to erase unwanted drawings.</li>
	<li>Click on the Measurements menu at the top of the image window to create a measurement protocol; a window containing different tools opens.</li>
	<li>Drag Find objects to top of the window (pane). Give a descriptive name to the Population&#160;1, select the associated color, and click on Measure. Another window opens.</li>
	<li>Choose a parameter i.e., volume or surface area. Click on OK. The data will appear in a table.</li>
	<li>To save the data click on the Measurement menu and select Make Measurement Item.</li>
	<li>Select A new measurement item called from the opened window. Then give a name to the data and click on OK.</li>
	<li>Finally, export the data to statistics software such as Graph pad or excel program. Use t-test to analyze the data, then plot 2D graphs.</li>
</ol>

<ol><li>Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+mystery+and+magic+of+glia%3A+a+perspective+on+their+roles+in+health+and+disease%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">The mystery and magic of glia: a perspective on their roles in health and disease.</a> Neuron. 60 (3), 430-440 (2008).</li>
<li>Pellerin, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activity-dependent+regulation+of+energy+metabolism+by+astrocytes%3A+an+update%5BTitle%5D)+AND+%22Glia%22%5BJournal%5D)">Activity-dependent regulation of energy metabolism by astrocytes: an update.</a> Glia. 55 (12), 1251-1262 (2007).</li>
<li>Sofroniew, M. V., Vinters, H. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Astrocytes%3A+biology+and+pathology%5BTitle%5D)+AND+%22Acta+Neuropathol%22%5BJournal%5D)">Astrocytes: biology and pathology.</a> Acta Neuropathol. 119 (1), 7-35 (2010).</li>
<li>Pekny, M., Nilsson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Astrocyte+activation+and+reactive+gliosis%5BTitle%5D)+AND+%22Glia%22%5BJournal%5D)">Astrocyte activation and reactive gliosis.</a> Glia. 50 (4), 427-434 (2005).</li>
<li>Sofroniew, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+dissection+of+reactive+astrogliosis+and+glial+scar+formation%5BTitle%5D)+AND+%22Trends+Neurosci%22%5BJournal%5D)">Molecular dissection of reactive astrogliosis and glial scar formation.</a> Trends Neurosci. 32 (12), 638-647 (2009).</li>
<li>Anderova, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+death%2Fproliferation+and+alterations+in+glial+morphology+contribute+to+changes+in+diffusivity+in+the+rat+hippocampus+after+hypoxia-ischemia%5BTitle%5D)+AND+%22J+Cereb+Blood+Flow+Metab%22%5BJournal%5D)">Cell death/proliferation and alterations in glial morphology contribute to changes in diffusivity in the rat hippocampus after hypoxia-ischemia.</a> J Cereb Blood Flow Metab. 31 (3), 894-907 (2011).</li>
<li>Hatten, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuronal+regulation+of+astroglial+morphology+and+proliferation+in+vitro%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Neuronal regulation of astroglial morphology and proliferation in vitro.</a> J Cell Biol. 100 (2), 384-396 (1985).</li>
<li>Paxinos, G., Watson, C. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPaxinos%2C+G+%22A+stereotaxic+atlas+of+the+rat+brain%22">A stereotaxic atlas of the rat brain</a>. , Academic. New York. (1998).</li>
<li>Kirby, E. D., Jensen, K., Goosens, K. A., Kaufer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stereotaxic+surgery+for+excitotoxic+lesion+of+specific+brain+areas+in+the+adult+rat%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Stereotaxic surgery for excitotoxic lesion of specific brain areas in the adult rat.</a> J Vis Exp. (65), e4079(2012).</li>
<li>Bagheri, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Amyloid+beta%281-40%29-induced+astrogliosis+and+the+effect+of+genistein+treatment+in+rat%3A+a+three-dimensional+confocal+morphometric+and+proteomic+study%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">Amyloid beta(1-40)-induced astrogliosis and the effect of genistein treatment in rat: a three-dimensional confocal morphometric and proteomic study.</a> PloS One. 8 (10), e76526(2013).</li>
<li>Chvatal, A., Anderova, M., Kirchhoff, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Three-dimensional+confocal+morphometry+-+a+new+approach+for+studying+dynamic+changes+in+cell+morphology+in+brain+slices%5BTitle%5D)+AND+%22J+Anat%22%5BJournal%5D)">Three-dimensional confocal morphometry - a new approach for studying dynamic changes in cell morphology in brain slices.</a> J Anat. 210 (6), 671-683 (2007).</li>
<li>Kulkarni, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+3-D+analysis+of+GFAP+labeled+astrocytes+from+fluorescence+confocal+images%5BTitle%5D)+AND+%22J+Neuroscie+Methods%22%5BJournal%5D)">Quantitative 3-D analysis of GFAP labeled astrocytes from fluorescence confocal images.</a> J Neuroscie Methods. 15 (246), 38-51 (2015).</li>
<li>Wagner, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Object-based+analysis+of+astroglial+reaction+and+astrocyte+subtype+morphology+after+ischemic+brain+injury%5BTitle%5D)+AND+%22Acta+Neurobiol+Exp%22%5BJournal%5D)">Object-based analysis of astroglial reaction and astrocyte subtype morphology after ischemic brain injury.</a> Acta Neurobiol Exp. 73 (1), 79-87 (2013).</li>
</ol>

This work was supported by grants from the County Council of &#214;sterg&#246;tland, (Sweden) and the Cellular and Molecular Research Center at Iran University of Medical Sciences (Tehran, Iran).

In the current protocol, we employed 3D confocal morphometry to evaluate 12 different parameters that were associated with astrocyte morphology. For this purpose, hippocampal tissue of rats with A&#946;1-40&#8210;induced astrogliosis, with or without genistein pretreatment as an anti-inflammatory agent were used. By using 3D images and morphometric software, we were able to show the effect of genistein on astrogliosis i.e. the morphology of astrocytes.
Changes in...

In healthy central nervous system (CNS), astrocytes play an important role in the regulation of blood flow, energy metabolism, synaptic function and plasticity, and extracellular ion and neurotransmitter homeostasis1-3. In addition, astrocytes respond to different harmful stimuli and abnormal conditions such as trauma, infection, ischemia or neurodegeneration via reactive astrogliosis which is characterized by hypertrophy, proliferation and functional remodeling of astrocytes4,5.
Reactive astrogliosis can engineer the inflammatory response and repair process in the tissue and, therefore, can affect the clinical outcome of therapeutic interventions. Accordingly, astrocytes have received attention from neuroscientist during the last decades as potential targets for therapeutic interventions for a variety of diseases affecting the CNS.
Astrocytes normally have a stellate shape with well-defined branches that spread around the soma6. In a diseased condition in the brain, astrocyte branches become convoluted and show swollen ends7, for example in the presence of amyloid beta (A&#946;).
This article presents a protocol for analyzing 3D images of astrocytes acquired by confocal microscopy. Twelve different quantitative parameters for each astrocyte were measured: the surface areas and volumes of the astrocyte territory (the tissue covered by an astrocyte), entire cell (including branches), cell body, and nucleus; the total length and number of branches; the fluorescence intensity of antibodies used for astrocyte detection; and the density of astrocytes (number/1,000 &#181;m2). For this purpose, we used brain sections from rats exposed to intrahippocampal injection of A&#946;1-40 with or without genistein treatment as an anti-inflammatory substance. The described protocol can be used for morphometric analysis of different cell types in vitro or in vivo in different conditions.

<table><tbody><tr><td>Amyloid beta 1-40</td><td>Sigma Aldrich</td><td>79793</td><td>Keep in -70 &amp;deg;C</td></tr><tr><td>Genistein</td><td>Sigma Aldrich</td><td>446-72-0</td><td>keep in -20 &amp;deg;C</td></tr><tr><td>polycolonal rabbit antibodies against glial fibrillary acidic protein</td><td>DAKO</td><td>Z0334</td><td></td></tr><tr><td>alkaline phosphate-conjugated swine anti-rabbit IgG antibodies</td><td>DAKO</td><td></td><td></td></tr><tr><td>Liquid Permanent Chromogen</td><td>DAKO</td><td>K0640</td><td></td></tr><tr><td>Liquid permanent Red Substrate Buffer</td><td>DAKO</td><td>K0640</td><td></td></tr><tr><td>Cremophor EL</td><td>Sigma Aldrich</td><td>27963</td><td>Polyethoxylated castor oil - Step 1.1</td></tr><tr><td>LSM 700 Confocal Laser Scanning Microscopy</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Volocity 6.3</td><td>Perkin Elmer Inc.,</td><td></td><td></td></tr><tr><td>Image Analysis 2000</td><td>Tekno Optic</td><td></td><td></td></tr><tr><td>Streotaxic apparatus</td><td>Stoelting</td><td></td><td></td></tr><tr><td>Graph pad Prism 5</td><td>Graph pad software Inc.</td><td></td><td></td></tr></tbody></table>

protocol for three-dimensional confocal morphometric analysis of astrocytes

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.

This section presents some examples of the qualitative and quantitative observations produced by 3D morphometric analysis. For complete results of all 12 parameters mentioned earlier, please see our previous publication10.
Qualitative Observations
Astrocytes exhibited thin or thick branches that were usually long in Aβ1-40 injected rats (Figure 1). A few small stellate-shaped astrocytes with short branches were observed in the ...

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 ...

The overall goal of this procedure is to assess astrocyte morphology by analyzing three dimensional images of astrocytes that were acquired via confocal microscopy. This method can be used to evaluate morphological changes that can appear in various cell types, either in pathological conditions or as the effect of an intervention strategy. The main advantage of this technique is that many parameters associated with cellular architecture can be studied simultaneously.Dr.Mariam Bodel will demonstrate this procedure Following the preparation of slides for immunohistochemical staining de parize sections by submersing them in xylene two times for 10 minutes. Incubate the slides for 10 minutes in each of 99.8%95%and 70%ethanol to rehydrate the sections after washing. PBS incubate the slides in a one to 1500 dilution of glial fibrillary acidic protein or GFAP primary antibody solution at four degrees Celsius overnight.Following incubation, wash the sections in PB S3 times for five minutes. Then incubate the slides with a one to 100 alkaline phosphatase conjugated secondary antibody solution for one hour at room temperature. Again, wash the section in PB S3 times for five minutes, less tha

protocol-for-three-dimensional-confocal-morphometric-analysis

Research

JoVE Journal

Neuroscience

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

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.

Immunostaining of Fluorescently Labeled Astrocytes in a Mouse Brain Tissue Section

Source: Moye, S. L., et.al. Visualizing Astrocyte Morphology Using Lucifer Yellow Iontophoresis. J. Vis. Exp. (2019). 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...

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

Source: Bagheri, M., et. al. Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes. J. Vis. Exp. (2015).This video demonstrates the imaging of fluorescently labeled astrocytes in a mouse brain section using a confocal microscope. The brain section is positioned under the appropriate objective lens, and suitable fluorophores and filters are selected. After optimizing the exposure automatically, the scanning parameters are defined to ensure high-resolution 2D images. Z-stack...

Three-dimensional Imaging of Immunolabeled Astrocytes Using Confocal Microscopy

A way to gain new insights into the complexity of the brain inflammatory response is presented. We describe immunofluorescence-based protocols followed by three-dimensional confocal analysis to investigate the pattern of co-expression of microglia/macrophage phenotype markers in a mouse model of focal ischemia.

Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced pluripotent stem cells.

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

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.

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

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.

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

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.

Visualizing Astrocyte Morphology Using Lucifer Yellow Iontophoresis

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

Investigation of Spatial Interaction Between Astrocytes and Neurons in Cleared Brains

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.

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

Öğrenin, konfokal mikroskopi ile astrosit morfolojisini üç boyutlu analiz etmeyi. Bu yöntem, patolojik koşullarda veya terapötik müdahaleler sonrası hücresel mimari değişikliklerini değerlendirmeye yardımcı olur.

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

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