The authors would like to thank Matthew Florence for preparation of the hippocampal slice cultures. This work was supported by grants from US National Institute on Aging and Alzheimer's Association to NZG.

1. Fixation
Fixatives are carcinogenic; wear gloves and handle the fixatives in a fume hood. Unless otherwise noted, all incubations are done on ice and all solutions should be filtered before use. Use electron microscopy-grade reagents.
Day 1
<ol>
	<li class="jove_content">After experimental conditions (e.g. viral injection, drug treatment), place the membrane with organotypic hippocampal slices in a 60 x 15 mm polystyrene Petri dish containing ice-cold 0.1 M phosphate buffer (Sorensen's phosphate buffer), pH 7.3. Add 1 - 2 ml of 0.1 M phosphate buffer directly on top of the slices to keep them cold.</li>
</ol>
CRITICAL STEP: Always use freshly prepared buffers and fixatives. Make sure the pH of buffer is within desired range. A failure to do so could damage cellular structures.
<ol start="2">
	<li>To isolate the CA1 subfield of the slice, use a disposable scalpel to gently cut across the slice next to the DG, parallel to the CA1 cell layer (Figure 1). Then cut the remaining slice vertically to remove the CA3 subfield and the subiculum. Cut a corner of the slice to help identify the top surface of the tissue.</li>
</ol>
NOTE: In case no viral delivery of protein of interest is required, the entire tissue slice can be fixed after the media is removed with a gentle rinse of ice-cold buffer. This is then followed by cutting out the CA1.
CRITICAL STEP: Keep track of the topside of the tissue in order for proper sectioning of the grids later on.
<ol start="3">
	<li>Carefully remove the tissue from the membrane using the backside of the scalpel. Use a Pasteur pipette to gently transfer the tissue to a 12-well plate containing 0.1 M phosphate buffer.</li>
	<li>Remove the buffer in the well from the plate and add 500 &mu;l - 1 ml of ice-cold fixative (pH 7.3) comprised of the following: 0.1% picric acid, 1% paraformaldehyde, and 2.5% glutaraldehyde in 0.1 M phosphate buffer. Incubate for 2 hr at 4 &deg;C.</li>
</ol>
NOTE: Picric acid may be explosive when dry. Keep it wetted with water in a container tightly closed. Store in a dry and well-ventilated place.
<ol start="5">
	<li>Remove the fixative from the well and wash samples 3 times (20 min each) in 0.1 M phosphate buffer.</li>
	<li>Incubate for 40 min in 1% tannic acid (w/v) in 0.1 M maleate buffer, pH 6.0.</li>
	<li>Rinse twice (20 min each) in maleate buffer.</li>
	<li>Incubate for 40 min in 1% uranyl acetate (w/v) in maleate buffer in the dark. Uranyl acetate is sensitive to light and is radioactive. Cover the beaker with Parafilm when dissolving and store any unused buffer in dark at 4 &deg;C.</li>
	<li>Rinse twice (20 min each) in maleate buffer.</li>
	<li>Incubate for 20 min in 0.5% platinum chloride (w/v) in maleate buffer.</li>
	<li>Rinse twice (20 min each) in maleate buffer. Store the samples at 4 &deg;C until they are ready for further processing.</li>
</ol>
2. Dehydration and Embedding
Propylene oxide is carcinogenic. Avoid vapors by working with it in a fume hood. Use absolute, 100% pure ethanol that contains no trace of water. Dilute this absolute ethanol to produce different concentrations for dehydration.
Day 2
<ol>
	<li>Incubate for 5 min in 50% ethanol and then for 5 min in 70% ethanol.</li>
	<li>Incubate for 15 min in freshly prepared 1% p-phenylenediamine (PPD) in 70% ethanol.</li>
	<li>Rinse three times in 70% ethanol.</li>
	<li>Incubate for 5 min in 80% ethanol and then for 5 min in 95% ethanol.</li>
	<li>Incubate in 100% ethanol twice 5 min each.</li>
	<li>Prepare glass vials with screw caps and make sure they are clean and completely dry. Transfer slices to glass vials and label each vial simply and clearly.</li>
	<li>Incubate for 5 min in 1:1 ethanol:propylene oxide.</li>
	<li>Incubate in 100% propylene oxide twice 5 min each.</li>
	<li>Add resin (Epon) to each vial to make a 1:1 mixture with propylene oxide. Gently mix for 2 hr. Do not shake the vials too rigorously to prevent bubbles that could interfere with embedding.</li>
	<li>Add resin to make a 3:1 mixture with propylene oxide, and mix for 2 hr.</li>
	<li>Transfer to 100% resin and incubate O/N.</li>
</ol>
Day 3
<ol start="12">
	<li>Sandwich samples between strips of ACLAR plastic and cure for 24 hr at 60 &deg;C.</li>
</ol>
3. Sectioning and Mounting on Grids
Day 4
<ol>
	<li>Cut semi-thin (0.5 &mu;m) sections and stain with 1% toluidine blue + 1% borax to determine the correct orientation of samples.</li>
	<li>Cut ultrathin sections (60 nm) and mount on nickel grids, one section per grid.</li>
</ol>
4. Immunohistochemistry
All buffers and water should be filtered before use.
Day 5
<ol>
	<li>Place a drop (~50 &mu;l) of 1% Tween-20/phosphate buffer (T/PB), pH 7.5, on a piece of clean Parafilm on a flat surface. Gently pick up the grid with clean forceps by the edge and float it on the buffer, section down, and incubate for 10 min at RT.</li>
	<li>Drain excess liquid from the grid by placing section side up on to filter paper and then float on 50 mM glycine in T/PB for 15 min at RT.</li>
</ol>
CRITICAL STEP: To avoid excessive 'carry-over' of solutions from one solution droplet to the next during the incubations, drain excess liquid by gently touching the edge of the grid onto #1 filter paper between each solution change. Also remove any solution trapped between the arms of the EM forceps holding the grid by gently wicking with a sliver of filter paper between the forceps arms while ensuring the sections do not dry out completely.
<ol start="3">
	<li>Dry the grid with filter paper and then float on blocking solution containing 2.5% BSA and 2.5% serum from the animal of the secondary antibody in T/PB for 30 min at RT.</li>
	<li>Incubate with primary antibody (in T/PB) at RT or O/N at 4 &deg;C. The optimal time and temperature of incubation, as well as antibody concentration, need to be experimentally determined.</li>
	<li>Wash the grid three times (2 min each) with T/PB.</li>
	<li>Incubate with secondary antibody (1:20 anti-rabbit or anti-mouse coupled to 10 nm gold) for 1 hr at RT.</li>
	<li>Wash three times (2 min each) with T/PB.</li>
	<li>Postfix with 2% glutaraldehyde in T/PB for 5 min.</li>
	<li>Wash three times (2 min each) with T/PB and then three times (2 min each) with filtered water.</li>
	<li>Incubate with 2% uranyl acetate in water for 10 min.</li>
</ol>
While the grid is staining, prepare a CO2-free chamber by placing a piece of Parafilm in the center of a glass Petri dish. Then place 4-6 pellets of NaOH in the dish around the Parafilm to absorb CO2 in the air. Keep the top closed for a few minutes. Use a Pasteur pipette and quickly transfer a small volume of Reynold's lead citrate solution to the Parafilm in the glass Petri dish. Open the top just enough to insert the Pasteur pipette to minimize the re-introduction of CO2 into the chamber.
NOTE: To make Reynold's lead citrate solution, boil 100 ml of deionized water in a microwave oven and let cool in an airtight container. In a 50 ml volumetric flask with stopper, mix 1.33 g lead nitrate, 1.76 g sodium citrate and 30 ml of the boiled water by shaking vigorously for 1 min. Then shake intermittently for 30 min. To this cloudy solution, add 8 ml of 1 M sodium hydroxide and slowly invert the flask a few times. Solution will become clear. Bring the solution to 50 ml with the boiled water. Store the solution tightly sealed. If precipitate appears, discard and make a new one.
<ol start="11">
	<li>In three smaller beakers prepare warm, freshly boiled deionized water. Wash off the uranyl acetate by dipping the grid in the first beaker and gently swirl it around for 30 sec. Repeat this step in the other two beakers.</li>
	<li>Open the top of the glass Petri dish just enough to place the grid on the drop of Reynold's lead citrate solution. If possible, wear a mask while doing this to avoid breathing on lead citrate and to prevent formation of precipitate. Incubate for 10 min.</li>
	<li>Open the top just enough to remove the grid. Avoid breathing on the lead citrate. Wash the grid three times by dipping it sequentially in three beakers with warm, freshly boiled distilled water. Let the grid dry on a piece of filter paper, section up. The sample is now ready for the electron microscope.</li>
</ol>

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J., Rustioni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+to+optimize+post-embedding+single+and+double+staining+for+amino+acid+neurotransmitters.">Techniques to optimize post-embedding single and double staining for amino acid neurotransmitters.</a> Journal of Histochemistry &amp; Cytochemistry. 40, 1011-1020 (1992).</li><li>Berryman, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+tannic+acid+on+antigenicity+and+membrane+contrast+in+ultrastructural+immunocytochemistry.">Effects of tannic acid on antigenicity and membrane contrast in ultrastructural immunocytochemistry.</a> J. Histochem. Cytochem. 40, 845-857 (1992).</li><li>Akagi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+methods+for+ultracryotomy+of+CNS+tissue+for+ultrastructural+and+immunogold+analyses.">Improved methods for ultracryotomy of CNS tissue for ultrastructural and immunogold analyses.</a> J. Neurosci. Methods. 153, 276-282 (2006).</li><li>Stirling, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructual+localization+of+lysozyme+in+human+colon+eosinophils+using+the+protein+A-gold+technique:+effects+of+processing+on+probe+distribution.">Ultrastructual localization of lysozyme in human colon eosinophils using the protein A-gold technique: effects of processing on probe distribution.</a> J. Histochem. Cytochem. 37, 709-714 (1989).</li><li>Phend, K. D., Rustioni, A., Weinberg, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+osmium-free+method+of+epon+embedment+that+preserves+both+ultrastructure+and+antigenicity+for+post-embedding+immunocytochemistry.">An osmium-free method of epon embedment that preserves both ultrastructure and antigenicity for post-embedding immunocytochemistry.</a> Journal of Histochemistry &amp; Cytochemistry. 43, 283-292 (1995).</li><li>Zhong, L., Cherry, T., Bies, C. E., Florence, M. A., Gerges, N. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogranin+enhances+synaptic+strength+through+its+interaction+with+calmodulin.">Neurogranin enhances synaptic strength through its interaction with calmodulin.</a> Embo J. 28, 3027-3039 (2009).</li><li>Zhong, L., Kaleka, K. S., Gerges, N. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogranin+phosphorylation+fine-tunes+long-term+potentiation.">Neurogranin phosphorylation fine-tunes long-term potentiation.</a> Eur. J. Neurosci. 33, 244-250 (2011).</li><li>Zhong, L., Gerges, N. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogranin+targets+calmodulin+and+lowers+the+threshold+for+the+induction+of+long-term+potentiation.">Neurogranin targets calmodulin and lowers the threshold for the induction of long-term potentiation.</a> PloS one. 7, e41275 (2012).</li><li>Horisberger, M., Vauthey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labelling+of+colloidal+gold+with+protein.+A+quantitative+study+using+beta-lactoglobulin.">Labelling of colloidal gold with protein. A quantitative study using beta-lactoglobulin.</a> Histochemistry. 80, 13-18 (1984).</li><li>Bendayan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+the+potential+and+versatility+of+colloidal+gold+cytochemical+labeling+for+molecular+morphology.">A review of the potential and versatility of colloidal gold cytochemical labeling for molecular morphology.</a> Biotech. Histochem. 75, 203-242 (2000).</li><li>Racz, B., Weinberg, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+organization+of+coflin+in+dendritic+spines.">Spatial organization of coflin in dendritic spines.</a> J. Neuroscience. 138 (2), 447-456 (2006).</li><li>Posthuma, G., Slot, J. W., Geuze, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Usefulness+of+the+immunogold+technique+in+quantitation+of+a+soluble+protein+in+ultra-thin+sections.">Usefulness of the immunogold technique in quantitation of a soluble protein in ultra-thin sections.</a> J. Histochem. Cytochem. 35, 405-410 (1987).</li><li>Howell, K. E., Reuter-Carlson, U., Devaney, E., Luzio, J. P., Fuller, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+antigen,+one+gold?+A+quantitative+analysis+of+immunogold+labeling+of+plasma+membrane+5'-nucleotidase+in+frozen+thin+sections.">One antigen, one gold? A quantitative analysis of immunogold labeling of plasma membrane 5'-nucleotidase in frozen thin sections.</a> Eur. J. Cell Biol. 44, 318-327 .</li><li>Yokota, E., Kawaguchi, T., Kusaba, T., Niho, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Immunologic+analysis+of+families+with+complement+receptor+deficiency].">[Immunologic analysis of families with complement receptor deficiency].</a> Nihon. Rinsho. 46, 2040-2045 (1988).</li><li>Kehle, T., Herzog, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+protein-gold+complexes+and+cell+surfaces:+a+method+for+precise+quantitation.">Interactions between protein-gold complexes and cell surfaces: a method for precise quantitation.</a> Eur. J. Cell Biol. 45, 80-87 (1987).</li><li>Bozzola, J. R., Lonnie, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+2.">Ch. 2.</a> Electron Microscopy. 30, (1992).</li><li>Cho, K. O., Hunt, C. A., Kennedy, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rat+brain+postsynaptic+density+fraction+contains+a+homolog+of+the+Drosophila+discs-large+tumor+suppressor+protein.">The rat brain postsynaptic density fraction contains a homolog of the Drosophila discs-large tumor suppressor protein.</a> Neuron. 9, 929-942 (1992).</li><li>Hunt, C. A., Schenker, L. J., Kennedy, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PSD-95+is+associated+with+the+postsynaptic+density+and+not+with+the+presynaptic+membrane+at+forebrain+synapses.">PSD-95 is associated with the postsynaptic density and not with the presynaptic membrane at forebrain synapses.</a> J. Neurosci. 16, 1380-1388 (1996).</li><li>Gispen, W. H., Leunissen, J. L., Oestreicher, A. B., Verkleij, A. J., Zwiers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+localization+of+B-50+phosphoprotein:+the+(ACTH)-sensitive+protein+kinase+substrate+involved+in+rat+brain+polyphosphoinositide+metabolism.">Presynaptic localization of B-50 phosphoprotein: the (ACTH)-sensitive protein kinase substrate involved in rat brain polyphosphoinositide metabolism.</a> Brain Research. 328, 381-385 (1985).</li><li>Biewenga, J. E., Schrama, L. H., Gispen, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+phosphoprotein+B-50/GAP-43+in+neuronal+and+synaptic+plasticity.">Presynaptic phosphoprotein B-50/GAP-43 in neuronal and synaptic plasticity.</a> Acta Biochim. Pol. 43, 327-338 (1996).</li><li>Maunsbach, A. A., Bjorn, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+16.">Ch. 16.</a> Biomedical Electron Microscopy Illustrated Methods and Interpretations. , 383-426 (1999).</li></ol>

We have nothing to disclose.

In this protocol, we have adopted the Phend and Weinberg method for brain and spinal cord tissues to study dendritic spines in rat hippocampal slice cultures. Dendritic spines in the hippocampal CA3-CA1 area are delicate structures containing a vast variety of proteins that play important roles in regulating neuronal functions. The presented method provides a balanced approach for achieving enhanced antigenicity while maintaining good ultrastructural preservation (Figure 2A), permitting reasonable...

<table border="1">
 <tbody>
 <tr>
 <td>NAME OF REAGENT</td>
 <td>COMPANY</td>
 <td>CATALOG NUMBER</td>
 <td></td>
 </tr>
 <tr>
 <td>60 x 15 mm polystyrene Petri dish</td>
 <td>Falcon</td>
 <td>351007</td>
 <td></td>
 </tr>
 <tr>
 <td>Disposable scalpel</td>
 <td>EXELINT</td>
 <td>29552</td>
 <td></td>
 </tr>
 <tr>
 <td>Cell culture inserts</td>
 <td>Millipore</td>
 <td>PICM03050</td>
 <td></td>
 </tr>
 <tr>
 <td>10 nm Goat-anti-rabbit gold</td>
 <td>Electron Microscopy Sciences</td>
 <td>25108</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-Neurogranin antibody</td>
 <td>Millipore</td>
 <td>AB5620</td>
 <td></td>
 </tr>
 <tr>
 <td>100% Picric acid</td>
 <td>Electron Microscopy Sciences</td>
 <td>19550</td>
 <td></td>
 </tr>
 <tr>
 <td>96% Paraformaldehyde</td>
 <td>Acros Organics</td>
 <td>AC41678-0030</td>
 <td></td>
 </tr>
 <tr>
 <td>25% Glutaraldehyde (EM grade)</td>
 <td>Sigma</td>
 <td>G5882</td>
 <td></td>
 </tr>
 <tr>
 <td>Uranyl acetate</td>
 <td>Electron Microscopy Sciences</td>
 <td>22400</td>
 <td></td>
 </tr>
 <tr>
 <td>p-Phenylenediamine</td>
 <td>Sigma</td>
 <td>P6001</td>
 <td></td>
 </tr>
 <tr>
 <td>Platinum (IV) chloride</td>
 <td>Sigma</td>
 <td>379840</td>
 <td></td>
 </tr>
 <tr>
 <td>Tannic acid</td>
 <td>Electron Microscopy Sciences</td>
 <td>21710</td>
 <td></td>
 </tr>
 </tbody>
</table>

post-embedding immunogold labeling of synaptic proteins in hippocampal slice cultures

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as colloidal gold can reveal the localization and distribution of specific antigens in various tissues1. The two most widely used techniques are pre-embedding and post-embedding techniques. In pre-embedding immunogold-electron microscopy (EM) techniques, the tissue must be permeabilized to allow antibody penetration before it is embedded. These techniques are ideal for preserving structures but poor penetration of the antibody (often only the first few micrometers) is a considerable drawback2. The post-embedding labeling methods can avoid this problem because labeling takes place on sections of fixed tissues where antigens are more easily accessible. Over the years, a number of modifications have improved the post-embedding methods to enhance immunoreactivity and to preserve ultrastructure3-5.
Tissue fixation is a crucial part of EM studies. Fixatives chemically crosslink the macromolecules to lock the tissue structures in place. The choice of fixative affects not only structural preservation but also antigenicity and contrast. Osmium tetroxide (OsO4), formaldehyde, and glutaraldehyde have been the standard fixatives for decades, including for central nervous system (CNS) tissues that are especially prone to structural damage during chemical and physical processing. Unfortunately, OsO4 is highly reactive and has been shown to mask antigens6, resulting in poor and insufficient labeling. Alternative approaches to avoid chemical fixation include freezing the tissues. But these techniques are difficult to perform and require expensive instrumentation. To address some of these problems and to improve CNS tissue labeling, Phend et al. replaced OsO4 with uranyl acetate (UA) and tannic acid (TA), and successfully introduced additional modifications to improve the sensitivity of antigen detection and structural preservation in brain and spinal cord tissues7. We have adopted this osmium-free post-embedding method to rat brain tissue and optimized the immunogold labeling technique to detect and study synaptic proteins.
We present here a method to determine the ultrastructural localization of synaptic proteins in rat hippocampal CA1 pyramidal neurons. We use organotypic hippocampal cultured slices. These slices maintain the trisynaptic circuitry of the hippocampus, and thus are especially useful for studying synaptic plasticity, a mechanism widely thought to underlie learning and memory. Organotypic hippocampal slices from postnatal day 5 and 6 mouse/rat pups can be prepared as described previously8, and are especially useful to acutely knockdown or overexpress exogenous proteins. We have previously used this protocol to characterize neurogranin (Ng), a neuron-specific protein with a critical role in regulating synaptic function8,9 . We have also used it to characterize the ultrastructural localization of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII)10. As illustrated in the results, this protocol allows good ultrastructural preservation of dendritic spines and efficient labeling of Ng to help characterize its distribution in the spine8. Furthermore, the procedure described here can have wide applicability in studying many other proteins involved in neuronal functions.

Figure 2B shows an example of the distribution of endogenous Ng molecules in dendritic spines of CA1 hippocampal pyramidal neurons. Nickel grids with ultrathin (60 nm) tissues containing CA1 region of the hippocampus (as seen in Figure 2A) were covered in 1% T/PB, 50 mM glycine, then blocked with 2.5% BSA and 2.5% serum prior to incubation with anti-Ng antibody. After washing with T/PB, grids were then covered in anti-rabbit secondary antibody coupled to 10 nm gold. Finally, grids were w...

Watch this Scientific Journal Video about Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures at JoVE.com

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons.

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

post-embedding-immunogold-labeling-synaptic-proteins-hippocampal

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Neuroscience

17.0K ビュー.  Medical College of Wisconsin. タンパク質の局在や分布は、細胞機能を理解するための重要な情報を提供します。電子顕微鏡の優れた空間分解能（EM）は、所定の抗原に続く免疫組織の細胞内局在を決定するために使用できます。抗原性はEM研究で特に困難であった維持しながら、構造的完全性を維持し、中枢神経系（CNS）の組織について。ここでは、ラット海馬CA1錐体細胞におけるシ?...

ビデオ: Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Investigation of Synaptic Tagging/Capture and Cross-capture using Acute Hippocampal Slices from Rodents

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity is achieved in neurons within a specific time frame. These long-term plasticity-related processes are the leading candidate models to study the basis of memory formation and persistence at the cellular level. Both STC and cross-tagging involve two serial processes: (1) setting of the synaptic tag as triggered by a specific pattern of stimulation, and (2) synaptic capture, whereby the synaptic tag interacts with newly synthesized plasticity-related proteins (PRPs). Much of the understanding about the concepts of STC and cross-tagging arises from the studies done in CA1 region of the hippocampus and because of the technical complexity many of the laboratories are still unable to study these processes. Experimental conditions for the preparation of hippocampal slices and the recording of stable late-LTP/LTD are extremely important to study synaptic tagging/cross-tagging. This video article describes the experimental procedures to study long-term plasticity processes such as STC and cross-tagging in the CA1 pyramidal neurons using stable, long-term field-potential recordings from acute hippocampal slices of rats.

This video article describes experimental procedures to study long-term plasticity and its associative processes such as synaptic tagging, capture and cross-tagging in the CA1 pyramidal neurons using acute hippocampal slices from rodents.

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.