Name: serial block-face scanning electron microscopy (sbem) for the study of dendritic spines
Uploaded: 2021-10-02T14:30:00
Description: Watch this Scientific Journal Video about Serial Block-Face Scanning Electron Microscopy SBEM for the Study of Dendritic Spines at JoVE.com

SBEM imaging, light microscopy imaging and electron microscopy sample preparation were performed with the use of the equipment of Laboratory of Imaging Tissue and Function which serves as an imaging core facility at the Nencki Institute of Experimental Biology.
For preparation of Figure 1, the image of a mouse (Souris_02), and a vial from the https://smart.servier.com/ was used.
This work was supported by the National Science Centre (Poland) Grant Opus (UMO-2018/31/B/NZ4/01603) awarded to KR.

The research was performed in compliance with Nencki Institute guidelines and permission of the Local Ethical Committee. The studies were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC), Animal Protection Act of Poland and approved by the first Local Ethics Committee in Warsaw. All efforts were made to minimize the number of animals used and their suffering.
CAUTION: All procedures described below must be carried out in a laboratory fu...

<ol>
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There are many variations of the primary NCMIR method described by Deerinck in 201010. The basic principles remain the same but, depending on the type of material studied, slight changes are implemented. It was described previously that different resins can be used to embed specimens for SBEM and for example in the case of plants, Spurr&#39;s is the resin of choice due to its low viscosity that allows better infiltration through the cell walls22,<sup class="xref"...

Most of the excitatory synapses in the central nervous system are located on dendritic spines - small protrusions of a neuronal membrane. These protrusions form confined biochemical compartments that control intracellular signal transduction. Structural plasticity of dendritic spines and synapses is closely related to the functional changes in synaptic efficacy that underlie such important processes as learning and memory1,2. It is important to note that electron microscopy (EM) is the only technique that allows to determine if a dendritic spine has a presynaptic input. EM resolution is also needed to study ultrastructural details such as shape of a postsynaptic density (PSD), representing a postsynaptic part of a synapse, or dimensions of a dendritic spine, as well as the size and shape of an axonal bouton. Additionally, with EM it is possible to visualize synapses and their surroundings.
Thanks to advances in imaging and computing technologies it is possible to reconstruct entire neural circuits. Volume electron microscopy techniques, such as serial section transmission electron microscopy (ssTEM), serial block-face scanning electron microscopy (SBEM), and focused ion beam scanning electron microscopy (FIB-SEM) are commonly used for neuronal circuit reconstructions3.
In our studies, the SBEM method is successfully employed to investigate the structural plasticity of dendritic spines and PSDs in samples of the mouse hippocampus and organotypic brain slices 4,5. The SBEM is based on the installation of a miniature ultramicrotome inside the scanning electron microscope chamber6,7,8,9. The top of the sample block is imaged, and then the sample is cut at a specified depth by the ultramicrotome, revealing a new block-face, which is again imaged and then the process is repeated8. As a result, only the image of a block-face is left while the slice which has been cut is lost as debris. That is why SBEM is called a destructive technique, meaning it is not possible to image the same place again. However, the advantage of the destructive on-block methods is that they do not suffer from warping problems and section loss that can significantly affect the data quality and the data analysis3. Moreover, SBEM gives the possibility to image a relatively large field of view ( &#62; 0.5 mm &#215; 0.5 mm) at high resolution3.
To employ SBEM, samples have to be prepared according to a dedicated, highly contrasting protocol due to the backscattered electron detector used for acquiring images. We show here how to perform sample preparation according to the protocol based on a procedure developed by Deerinck10 (National Center for Microscopy and Imaging Research (NCMIR)&#160;method), using reduced osmium-thiocarbohydrazide-osmium (rOTO) stains developed in the 1980s8,11. In addition, we introduce a two-step fixation approach, with mild fixation perfusion that allows to use the same brain both for immunofluorescence studies with light microscopy and SBEM.
In the protocol a mouse brain is primarily fixed with a mild fixative, and then cut into halves, and one hemisphere is postfixed and prepared for immunofluorescence (IF), while the other for EM studies (Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62712/62712fig01.jpg" /> 
Figure 1. Schematic representation of the workflow for the dendritic spines preparation for the analysis with SBEM. Mice were sacrificed and perfused with a mild primary fixative. The brain was cut into halves, and one hemisphere was postfixed with immunofluorescence (IF)-dedicated fixative, cryoprotected, sliced using a cryostat and processed for IF studies, while the other hemisphere was postfixed with EM fixative, sliced with the vibratome and prepared for EM studies. Brain slices for SBEM studies were contrasted, flat embedded in resin, then a CA1 region of the hippocampus was mounted to the pin, and imaged with SBEM (Figure 1). The part of the protocol highlighted in a yellow box was featured in the video. <a href="https://www.jove.com/files/ftp_upload/62712/62712fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anesthetic:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine/xylazine mixture (Ketamina/Sedazin)</td>
			<td>Biowet Pulawy, Pulawy, Poland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pentobarbital (Morbital)</td>
			<td>Biowet Pulawy, Pulawy, Poland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fixatives:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde (GA)</td>
			<td>Sigma-Aldrich,St. Louis, MI, USA</td>
			<td>G5882</td>
			<td>Grade I, 25% in H2O, specially purified for use as an electron microscopy fixative</td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl)</td>
			<td>POCH, Gliwice, Poland</td>
			<td>575283115</td>
			<td>pure p.a.</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma-Aldrich,St. Louis, MI, USA</td>
			<td>441244</td>
			<td>prilled, 95%</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS), pH 7.4</td>
			<td>Sigma-Aldrich,St. Louis, MI, USA</td>
			<td>P4417-50TAB</td>
			<td>tablets</td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Sigma-Aldrich,St. Louis, MI, USA</td>
			<td>S5881</td>
			<td>reagent grade, <img alt="Equation" src="/files/ftp_upload/62712/62712symbol.png" />98%, pellets (anhydrous)</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic (Na2HPO4)</td>
			<td>Sigma-Aldrich,St. Louis, MI, USA</td>
			<td>S3264</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic (NaH2PO4)</td>
			<td>Sigma-Aldrich,St. Louis, MI, USA</td>
			<td>S3139</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Large blunt/blunt curved scissors (~14.5 cm)</td>
			<td>Fine Science Tools, Foster City, CA, USA</td>
			<td>14519-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-spatula (double 2&#34; flat ends, one rounded, one tapered to 1/8&#34;)</td>
			<td>Fine Science Tools, Foster City, CA, USA</td>
			<td>10091-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle tip, 15 GA, blunt (perfusion needle)</td>
			<td>KD Medical GmbH Hospital Products, Berlin, Germany</td>
			<td>KD-FINE 900413</td>
			<td>1.80 x 40 mm</td>
		</tr>
		<tr>
			<td>Pair of fine (Graefe) tweezers</td>
			<td>Fine Science Tools, Foster City, CA, USA</td>
			<td>11050-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion pump</td>
			<td>Lead Fluid</td>
			<td>BQ80S</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic vials</td>
			<td>Profilab, Warsaw, Poland</td>
			<td>534.02</td>
			<td>plastic vials with blue cap for tissue storage, 20 ml, 31 x 48 mm</td>
		</tr>
		<tr>
			<td>Straight iris scissors (~9 cm)</td>
			<td>Fine Science Tools, Foster City, CA, USA</td>
			<td>14058-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain slices preparation for EM:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>12-well plate</td>
			<td>NEST, Rahway, NJ, USA</td>
			<td>712001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanoacrylic glue</td>
			<td>Fenedur, Montevideo, Uruguay</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass vials</td>
			<td>Electron Microscopy Sciences, Hatfield, PA, USA</td>
			<td>72632</td>
			<td>20 ml Scintillation Vial, a pack of 100</td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>VWR, Radnor, PA, USA</td>
			<td>612-4545</td>
			<td>LDPE, disposable, 7.5 ml</td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Wilkinson Sword, London, UK</td>
			<td></td>
			<td>Classic double edge safety razor blades</td>
		</tr>
		<tr>
			<td>Scalpel blade</td>
			<td>Swann-Morton, Sheffield, UK</td>
			<td>No. 20</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica Microsystems, Vienna, Austria</td>
			<td>Leica VT1000 S</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain slices preparation for IF:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>NEST, Rahway, NJ, USA</td>
			<td>701101</td>
			<td></td>
		</tr>
		<tr>
			<td>Criostat</td>
			<td>Leica Microsystems, Vienna, Austria</td>
			<td>Leica CM 1950</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene glycol</td>
			<td>Bioshop, Burlington, Canada</td>
			<td>ETH001</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-profile disposable blade 819</td>
			<td>Leica Biosystems Inc., USA</td>
			<td>14035838925</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blade</td>
			<td>Swann-Morton, Sheffield, UK</td>
			<td>No. 20</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide (NaN3)</td>
			<td>POCH, Gliwice, Poland</td>
			<td>792770426</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>POCH, Gliwice, Poland</td>
			<td>772090110</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue freezing medium for cryosectioning, OCT-Compound</td>
			<td>Leica Biosystems, Switzerland</td>
			<td>14020108926</td>
			<td></td>
		</tr>
		<tr>
			<td>Immunostaining:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>NEST, Rahway, NJ, USA</td>
			<td>702001</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Post Synaptic Density Protein 95 Antibody</td>
			<td>Merck-Millipore, Burlington, MA, USA</td>
			<td>MAB1598</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss, G&#246;ttingen, Germany</td>
			<td></td>
			<td>Zeiss Spinning Disc microscope (63 &#215; oil objective, NA 1.4, pixel size 0.13 &#181;m &#215; 0.13 &#181;m)</td>
		</tr>
		<tr>
			<td>Cover slide</td>
			<td>Menzel Glaser, Braunschweig, Germany</td>
			<td>B-1231</td>
			<td>24 x 60 mm</td>
		</tr>
		<tr>
			<td>Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555</td>
			<td>Invitrogen, Carlsbad, CA, USA</td>
			<td>A31570</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount-G Mounting Medium, with DAPI</td>
			<td>Invitrogen, Carlsbad, CA, USA</td>
			<td>00-4959-52</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slide</td>
			<td>Thermo Scientific, Waltham, MA, USA</td>
			<td>AGAA00008</td>
			<td>SuperFrost</td>
		</tr>
		<tr>
			<td>Normal donkey serum (NDS)</td>
			<td>Jackson ImmunoResearch Laboratories, West Grove, PA, USA</td>
			<td>017-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>JWElectronic, Warsaw, Poland</td>
			<td>KL-942</td>
			<td></td>
		</tr>
		<tr>
			<td>TritonT X-100 Reagent Grade</td>
			<td>Bioshop, Burlington, Canada</td>
			<td>TRX506</td>
			<td></td>
		</tr>
		<tr>
			<td>Electron microsocpy sample preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium hexacyanoferrate(II) trihydrate</td>
			<td>POCH, Gliwice, Poland</td>
			<td>746980113</td>
			<td></td>
		</tr>
		<tr>
			<td>Aclar 33C Film</td>
			<td>Electron Microscopy Sciences, Hatfield, PA, USA</td>
			<td>50425</td>
			<td>Fluoropolymer Film embedding sheet</td>
		</tr>
		<tr>
			<td>DMP-30, 2,4,6-Tris(dimethylaminomethyl)phenol</td>
			<td>Sigma-Aldrich,St. Louis, MI, USA</td>
			<td>T58203</td>
			<td>Epoxy embedding medium accelerator</td>
		</tr>
		<tr>
			<td>Durcupan ACM single component A, M</td>
			<td>Sigma-Aldrich,St. Louis, MI, USA</td>
			<td>44611</td>
			<td>Durcupan ACM single component A, M epoxy resin</td>
		</tr>
		<tr>
			<td>Durcupan ACM single component B</td>
			<td>Sigma-Aldrich,St. Louis, MI, USA</td>
			<td>44612</td>
			<td>Durcupan ACM single component B, hardener 964</td>
		</tr>
		<tr>
			<td>Durcupan ACM single component D</td>
			<td>Sigma-Aldrich,St. Louis, MI, USA</td>
			<td>44614</td>
			<td>Durcupan ACM single component D , plasticizer</td>
		</tr>
		<tr>
			<td>Ethyl alcohol absolute</td>
			<td>POCH, Gliwice, Poland</td>
			<td>64-17-5</td>
			<td>Ethyl alcohol absolute 99.8 % pure P.A.-BASIC</td>
		</tr>
		<tr>
			<td>Genlab laboratory oven</td>
			<td>Wolflabs, York, UK</td>
			<td>Mino/18/SS</td>
			<td>Oven Genlab MINO/18/SS 18l volume, no fan circulation, no digital display, standard temperature gradient, standard recovery rate, no timer, 250&#176;C maximum temperature, 240V electrical supply</td>
		</tr>
		<tr>
			<td>L-Aspartic acid</td>
			<td>Sigma-Aldrich,St. Louis, MI, USA</td>
			<td>A-9256</td>
			<td>reagent grade, <img alt="Equation" src="/files/ftp_upload/62712/62712symbol.png" />98% (HPLC)</td>
		</tr>
		<tr>
			<td>Lead (II) nitrate</td>
			<td>Sigma-Aldrich,St. Louis, MI, USA</td>
			<td>467790</td>
			<td><img alt="Equation" src="/files/ftp_upload/62712/62712symbol.png" />99.95% trace metals basis</td>
		</tr>
		<tr>
			<td>Osmium tetroxide</td>
			<td>Sigma-Aldrich,St. Louis, MI, USA</td>
			<td>75632</td>
			<td>for electron microscopy, 4% in H2O</td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Elmetron, Zabrze, Poland</td>
			<td>CP-5-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotator</td>
			<td>BioSan, J&#243;zef&#243;w, Poland</td>
			<td>Multi Bio RS-24</td>
			<td>rotator Multi Bio RS-24</td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Sigma-Aldrich,St. Louis, MI, USA</td>
			<td>S5881</td>
			<td>reagent grade, <img alt="Equation" src="/files/ftp_upload/62712/62712symbol.png" />98%, pellets (anhydrous)</td>
		</tr>
		<tr>
			<td>Sunflower mini shaker</td>
			<td>Grant bio, Shepreth,UK</td>
			<td>PD-3D</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filter</td>
			<td>Millipore, Burlington, MA, USA</td>
			<td>SLGP033NB</td>
			<td>0,22 &#181;m pore size</td>
		</tr>
		<tr>
			<td>Thiocarbohydrazide</td>
			<td>Sigma-Aldrich,St. Louis, MI, USA</td>
			<td>88535</td>
			<td>purum p.a., for electron microscopy, <img alt="Equation" src="/files/ftp_upload/62712/62712symbol.png" />99.0% (N)</td>
		</tr>
		<tr>
			<td>Uranyl acetate</td>
			<td>Serva, Heidelberg, Germany</td>
			<td>77870</td>
			<td>Uranyl acetate&#183;2H2O, research grade</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>WSL, Swietochlowice, Poland</td>
			<td>LWT</td>
			<td></td>
		</tr>
		<tr>
			<td>Specimen mounting for SBEM</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well culture plate</td>
			<td>VWR, Radnor, PA, USA</td>
			<td>734-2782</td>
			<td>96-well plates, round bottom, non treated</td>
		</tr>
		<tr>
			<td>AM Gatan 3View stub handling tweezers</td>
			<td>Micro to Nano, Haarlem, Netherlands 
			Netherlands</td>
			<td>50-001521</td>
			<td></td>
		</tr>
		<tr>
			<td>Binocular</td>
			<td>OPTA-TECH, Warsaw, Poland</td>
			<td>X2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Conductive glue</td>
			<td>Chemtronics, Georgia, USA</td>
			<td>CW2400</td>
			<td>conductive eopxy</td>
		</tr>
		<tr>
			<td>Gatan 3View sample pin stubs</td>
			<td>Micro to Nano, Haarlem, Netherlands 
			Netherlands</td>
			<td>10-006003</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Sigma-Aldrich,St. Louis, MI, USA</td>
			<td>P7793</td>
			<td>roll size 20&#160;in. &#215; 50&#160;ft</td>
		</tr>
		<tr>
			<td>Pelco conductive silver paint</td>
			<td>Ted Pella, Redding, CA, USA</td>
			<td>16062-15</td>
			<td>PELCO&#174;&#160;Conductive Silver Paint, 15g</td>
		</tr>
		<tr>
			<td>Razor blades double edge</td>
			<td>Electron Microscopy Sciences, Hatfield, PA, USA</td>
			<td>72000</td>
			<td>Stainless Steel &#34;PTFE&#34; coated. PERSONNA brand .004&#34; thick, wrapped individually, 250 blades in a box.</td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope</td>
			<td>Zeiss, Oberkochen, Germany</td>
			<td></td>
			<td>Sigma VP with Gatan 3View2 chamber, acceleration voltage 2.5 kV, variable pressure 5 Pa, aperture 20 &#181;m, dwell time 6 &#181;s, slice thickness 60 nm, magnification 15 000 x, image resolution 2048 x 2048 pixels, pixel size 7.3 nm</td>
		</tr>
		<tr>
			<td>trim 90&#176; diamond knife</td>
			<td>Diatome Ltd., Nidau, Switzerland</td>
			<td>DTB90</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultramicrotome</td>
			<td>Leica Microsystems, Vienna, Austria</td>
			<td>Leica ultracutR</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td>webpage</td>
			<td></td>
			<td>tutorials</td>
		</tr>
		<tr>
			<td>FijiJ</td>
			<td>https://fiji.sc/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscopy Image Browser</td>
			<td>http://mib.helsinki.fi/</td>
			<td></td>
			<td>http://mib.helsinki.fi/tutorials.html</td>
		</tr>
		<tr>
			<td>Reconstruct</td>
			<td>https://synapseweb.clm.utexas.edu/software-0</td>
			<td></td>
			<td>https://synapseweb.clm.utexas.edu/software-0)</td>
		</tr>
		<tr>
			<td>Animals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>Adult 3-month old and&#160;20&#177;1&#160;month old&#160;female&#160;Thy1-GFP(M) mice (Thy1-GFP +/-) (Feng et al.,2000) which express GFP in a sparsely distributed population of glutamatergic neurons. Animals were bred as heterozygotes with the C57BL/6J background in the Animal House of the Nencki Institute of Experimental Biology.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

serial block-face scanning electron microscopy (sbem) for the study of dendritic spines

Three-dimensional electron microscopy (3D EM) gives a possibility to analyze morphological parameters of dendritic spines with nanoscale resolution. In addition, some features of dendritic spines, such as volume of the spine and post-synaptic density (PSD) (representing post-synaptic part of the synapse), presence of presynaptic terminal, and smooth endoplasmic reticulum or atypical form of PSD (e.g., multi-innervated spines), can be observed only with 3D EM. By employing serial block-face scanning electron microscopy (SBEM) it is possible to obtain 3D EM data easier and in a more reproducible manner than when performing traditional serial sectioning. Here we show how to prepare mouse hippocampal samples for SBEM analysis and how this protocol can be combined with immunofluorescence study of dendritic spines. Mild fixation perfusion allows us to perform immunofluorescence studies with light microscopy on one half of the brain, while the other half was prepared for SBEM. This approach reduces the number of animals to be used for the study.

Using the method described above high contrast, good resolution images of the mouse brain tissue can be obtained. A large field of view provided by the SBEM technique facilitates precise selection of the region of interest. The large image of the CA1 region of the hippocampus was taken to measure the length of stratum radiatum (SR) (Figure 2A) and to set the imaging precisely in the center (Figure 2B). Next, the stack of images was acquired and the obje...

Watch this Scientific Journal Video about Serial Block-Face Scanning Electron Microscopy SBEM for the Study of Dendritic Spines at JoVE.com

Serial Block-Face Scanning Electron Microscopy SBEM for the Study of Dendritic Spines

Serial Block-Face Scanning Electron Microscopy (SBEM) is applied to image and analyze dendritic spines in the murine hippocampus.

Serial Block-Face Scanning Electron Microscopy (SBEM) for the Study of Dendritic Spines

Three-dimensional electron microscopy (3D EM) gives a possibility to analyze morphological parameters of dendritic spines with nanoscale resolution. In ...

Research

JoVE Journal

Neuroscience

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

Focussed Ion Beam Milling and Scanning Electron Microscopy of Brain Tissue

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron ...

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Correlative Light and Electron Microscopy to Study Microglial Interactions with &#946;-Amyloid Plaques

Correlative Light and Electron Microscopy to Study Microglial Interactions with ÃƒÅ½Ã‚Â²-Amyloid Plaques

A detailed protocol is provided here to identify amyloid A&#946; plaques in brain sections from Alzheimer&#39;s disease mouse models before pre-embedding ...

Analysis of Brain Mitochondria Using Serial Block-Face Scanning Electron Microscopy

Human brain is a high energy consuming organ that mainly relies on glucose as a fuel source. Glucose is catabolized by brain mitochondria via glycolysis, ...