This work was supported by National Institutes of Health NINDS R01 NS105005 (KRG) and NS105005-03S1 (KRG), Department of Defense USAMRMC W81XWH-14-1-0061 (KRG), NIAAA R01AA016852, NIAAA T32AA007565 (CFH), and&#160;a grant from FRAXA Research (CFH) and the Alzheimer&#39;s Association, AARG-NTF-21-852843 (KRG), AARF-19-614794-RAPID (KRG).&#160;

Isolation of cells and tissue from animals was in accordance with the National Institutes of Health&#39;s Guide for the Care and Use of Laboratory Animals and approved by the Wake Forest Institutional Animal Care and Use Committee
NOTE: This protocol is used on samples already treated and fixed per specific experimental paradigms and requirements. For demonstration purposes, synapse formation due to rapid antidepressant treatment is used to highlight this synapse detection technique6. Neurons previously cultured on coverslips, treated, fixed in 4% paraformaldehyde (PFA), and stored in 1x phosphate-buffered saline (PBS) will be used to highlight the in vitro procedures. Previously sliced hippocampal tissue (25 &#181;m thick) from mice treated, transcardially perfused with ice-cold PBS and 4% PFA, and then stored in cryoprotectant will be used to highlight the slice procedures. Please see11,12 for more information about how to culture neurons or transcardially perfuse rodents. See Figure 1 for a graphical representation of this procedure.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63139/63139fig01.jpg" /> 
Figure 1: Graphical representation of DetectSyn assay. After permeabilizing cell membranes, primary antibodies for Synapsin1 and PSD95 bind to these synaptic proteins. Secondaries with oligonucleotide tags then bind to the primary antibodies. If Synapsin1 and PSD95 are within 40 nm, as at a synapse, then the oligonucleotides interact, and a fluorescent tag is amplified. This fluorescent signal can then be imaged via standard microscopy and analyzed. <a href="https://www.jove.com/files/ftp_upload/63139/63139fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
1. Rinse samples
<ol>
	<li>Rinse the samples with 500 &#181;L of 1x PBS + 0.75% glycine for 5 min 3 times with gentle agitation on an orbital shaker to remove residual PFA or cryoprotectant.</li>
</ol>
2. Block and permeabilize samples
<ol>
	<li>Prepare blocking and permeabilization solution (10% normal donkey serum, 0.25% Tween 20) in 1x PBS. Prepare enough to use for blocking, primary, and secondary incubations.</li>
	<li>To samples (e.g., coverslips or free-floating slices) in 24 well plates, add 500 &#181;L of blocking and permeabilization solution. Ensure each well contains a different sample and is appropriately labeled to prevent samples from being switched.</li>
	<li>Incubate the samples at room temperature (RT) for 60 min for cultured cells or 2 h for sliced tissue. Use an orbital shaker for gentle agitation.</li>
</ol>
3. Incubate samples in primary antibodies
<ol>
	<li>Prepare primary antibodies in blocking buffer:
	<ol>
		<li>Prepare Postsynaptic density 95 (PSD95; 1:500, rabbit polyclonal), Synapsin1 (1:500, mouse monoclonal), MAP2 (1:400, chicken polyclonal)</li>
		<li>Prepare a negative control aliquot that omits one of the synaptic pairs (e.g., without PSD95)</li>
	</ol>
	</li>
	<li>Carefully remove the blocking solution with a plastic Pasteur pipette. Try to remove as much as possible without disturbing the cells or tearing tissue.</li>
	<li>For cultured cells:
	<ol>
		<li>Line a large plastic petri dish with parafilm. Carefully transfer coverslips to the parafilm using forceps.</li>
		<li>Carefully add 60 &#181;L of the primary antibody solution to the top of the coverslips. Make sure not to spill the primary antibody solution over the side of the coverslip.</li>
		<li>To provide humidity and prevent samples from drying out during the incubation period, add ultrapure water to a smaller Petri dish and carefully arrange the small Petri dish around the coverslips.</li>
		<li>Cover the large Petri dish and incubate cultured cells for 1 h at RT.</li>
	</ol>
	</li>
	<li>For sliced tissue:
	<ol>
		<li>Carefully add 250 &#181;L of the primary antibody solution to free-floating slices in a 24 well plate.</li>
		<li>Cover the plate and incubate the tissue overnight at 4 &#176;C with gentle agitation on an orbital shaker.</li>
	</ol>
	</li>
</ol>
4. Wash samples, then incubate in secondary antibodies
<ol>
	<li>Prepare secondary antibodies in the blocking buffer:
	<ol>
		<li>Prepare Donkey anti-mouse (1:5), donkey anti-rabbit (1:5), donkey anti-chicken (1:400).</li>
		<li>At this step, additional technical controls can be obtained by preparing a secondary aliquot that omits either the anti-mouse or anti-rabbit secondary.</li>
	</ol>
	</li>
	<li>For cultured cells:
	<ol>
		<li>Using forceps, carefully tap off the primary solution from coverslips onto a paper towel</li>
		<li>Using forceps, carefully transfer the coverslips back to their original 24 well plate filled with 500 &#181;L of 1x PBS.</li>
	</ol>
	</li>
	<li>For sliced tissue:
	<ol>
		<li>Carefully remove the primary antibody solution with a plastic Pasteur pipette. Try to remove as much as possible without tearing tissue.</li>
		<li>Add 500 &#181;L of 1x PBS</li>
	</ol>
	</li>
	<li>Wash the samples for 10 min 3 times in 1x PBS with gentle agitation on an orbital shaker. During this time, bring all wash buffers to RT.
	<ol>
		<li>During this time, change the parafilm in the large Petri dish</li>
	</ol>
	</li>
	<li>For cultured cells:
	<ol>
		<li>Using forceps, carefully transfer the coverslips back to the parafilmed large Petri dish</li>
		<li>Carefully add 40 &#181;L of the secondary antibody solution to the top of the coverslips. Make sure not to spill the secondary antibody solution over the side of the coverslip.</li>
		<li>If needed, add more ultrapure water to a smaller petri dish and carefully arrange the small petri dish around coverslips.</li>
		<li>Cover the large Petri dish</li>
	</ol>
	</li>
	<li>For sliced tissue:
	<ol>
		<li>Carefully add 250 &#181;L of the secondary antibody solution to free-floating slices in a 24 well plate.</li>
		<li>Cover the plate 
		NOTE: From here on, protect samples from light by wrapping the tops of the plates with foil.</li>
	</ol>
	</li>
	<li>Incubate the samples at 37 &#176;C for 1 h</li>
</ol>
5. Ligation
<ol>
	<li>Mix the ligation stock 1:5 in molecular-grade water.</li>
	<li>As in section 4, carefully transfer the coverslips and remove the secondary mix from the sliced tissue.</li>
	<li>Wash the samples in 500 &#181;L of wash buffer A
	<ol>
		<li>For cultured cells, wash 2 times for 5 min. For sliced tissue, wash 2 times for 10 min. Use gentle agitation on an orbital shaker for both.</li>
		<li>During this time, change the parafilm in the large Petri dish</li>
	</ol>
	</li>
	<li>While keeping the ligase on a cold block, dilute the ligase 1:40 in the ligation stock from step 5.1. Perform this dilution immediately before adding the ligase to the samples.</li>
	<li>As in section 4, remove as much of the wash buffer A as possible from samples before adding the ligase.</li>
	<li>For cultured cells: Transfer coverslips back to the parafilmed Petri dish. Add 40 &#181;L of the ligation mix to coverslips, arrange small water-filled Petri dishes around the coverslips, and cover the large Petri dish.</li>
	<li>For sliced tissue: Add 250 &#181;L of the ligation mix from Step 5.4 to each well and cover the plate.</li>
	<li>Incubate the samples for 30 min at 37 &#176;C.</li>
</ol>
6. Amplification
<ol>
	<li>Mix the amplification stock 1:5 in molecular-grade water.</li>
	<li>As in section 4, carefully transfer the coverslips and remove the ligation mix from the sliced tissue.</li>
	<li>Wash samples in 500 &#181;L of wash buffer A
	<ol>
		<li>For cultured cells, wash 2 times for 2 min. For sliced tissue, wash 2 times for 10 min. Use gentle agitation on an orbital shaker for both.</li>
		<li>During this time, change the parafilm in the large Petri dish</li>
	</ol>
	</li>
	<li>Perform this dilution immediately before adding the polymerase to samples. While keeping the polymerase on a cold block, dilute polymerase
	<ol>
		<li>For cultured cells, dilute polymerase 1:80 in the amplification stock from step 6.1.</li>
		<li>For sliced tissue, dilute polymerase 1:40 in the amplification stock from step 6.1.</li>
	</ol>
	</li>
	<li>As in step 4, remove as much of the wash buffer A as possible from samples before adding the polymerase.</li>
	<li>For cultured cells: Transfer the coverslips back to the parafilmed Petri dish. Add 40 &#181;L of the amplification mix from Step 6.4.1 to coverslips, arrange small water-filled Petri dishes around the coverslips, and cover the large Petri dish. Incubate the samples for 100 min at 37 &#176;C.</li>
	<li>For sliced tissue: Add 250 &#181;L of the amplification mix from Step 6.4.2 to each well and cover the plate. Incubate the samples for 2 h at 37 &#176;C. 
	NOTE: During this time, prepare and label slides.</li>
</ol>
7. Mounting
<ol>
	<li>As in Step 4, carefully transfer the coverslips and remove the amplification mix from the sliced tissue.</li>
	<li>Wash the samples in 500 &#181;L of wash buffer B 2 times for 10 min with gentle agitation on an orbital shaker.</li>
	<li>Wash the samples in 500 &#181;L of 1% wash buffer B for 1 min with gentle agitation on an orbital shaker.</li>
	<li>For cultured cells:
	<ol>
		<li>Drop 3 &#181;L of mounting media onto a slide</li>
		<li>Tap off excess wash buffer from the coverslip and then place the coverslip (with cells facing down) into the mounting media. Seal the sides with a small amount of clear nail polish to seal the coverslip in place.</li>
	</ol>
	</li>
	<li>For sliced tissue:
	<ol>
		<li>Carefully transfer a tissue slice to the prepared slide and arrange it, so the slice is lying flat. Drop between 5-10 &#181;L of mounting media (amount will depend on the size of the slice) onto tissue slice</li>
		<li>Carefully place a glass coverslip over tissue slice and seal with a small amount of clear nail polish along the edge to seal the coverslip in place.</li>
	</ol>
	</li>
	<li>Wait at least 15 min before analyzing under the microscope, or store at -20 &#176;C.</li>
</ol>
8. Obtain digital images with a confocal microscope
<ol>
	<li>Optimize the acquisition settings (e.g., laser power, gain, offset) across samples from all treatments. Ensure that the optimization includes decreasing background noise and enhancing the signal without oversaturating the intensity of the fluorescent signals. Once settings are determined, apply the same acquisition settings across all images obtained. 
	NOTE: The following acquisition details can be used with a Nikon A1 confocal microscope and the Nikon NIS AR Elements software.</li>
	<li>Set the slide with sample on the stage and find the focal plane for the sample using DAPI through the eyepiece.</li>
	<li>Turn off the eye port by clicking on Eye port and choose an optical configuration button to adjust the settings.</li>
	<li>Adjust the gain, offset, and laser power for each fluorescent channel to decrease background noise and enhance the fluorescent signal. Make sure the fluorescent signal does not become oversaturated as mentioned in steps 8.5-8.6.</li>
	<li>Monitor oversaturation using a pseudocolor for the fluorescent signal. At the bottom of the live image, right-click the tab labeled with the fluorescent channel currently being used.</li>
	<li>Next, choose Channel Coloring and pick a pseudocolor like Rainbow Dark to visualize the fluorescence intensity in a heat-map-like pseudocolor. In Rainbow Dark, cooler colors indicate less fluorescent intensity, and hotter colors indicate more fluorescent intensity.</li>
	<li>Once all fluorescent channels are optimized, right-click on the optical configuration button previously chosen and choose Assign Current Camera Setting for this button.</li>
	<li>Verify that the chosen settings are sufficient for a random sample from each treatment group. If the chosen settings oversaturate any of these samples, repeat step 8.4 to eliminate the oversaturation.</li>
	<li>For cultured neurons, follow steps 8.10-8.16.</li>
	<li>Using the eye-port, search for a neuron with dendrites that have minimal overlap with other dendrites.</li>
	<li>Turn off the eye port and use the DAPI channel to visualize the cell body of the chosen neuron. Double-click on the center of the soma to center the neuron in the middle of the field of view.</li>
	<li>Using the MAP2 channel, find the best plane of focus for the MAP2 signal with live scanning.</li>
	<li>Under the ND Acquisition tab, click on Save to File and choose a file to save the image into under Browse. Then, input the filename.</li>
	<li>Under the Z tab, select the Symmetric Mode Defined by Range option. Set the focus to the best MAP2 plane and click on the Relative button to set this focal plane as the middle of the z-stack.</li>
	<li>Set the range to 5 &#181;m with 1 &#181;m steps, and make sure to check Close Active Shutter During Z Movement. Under the Wavelength tab, select the name of the optical button with the previously configured acquisition settings under Optical Conf. Then, click on Run Now.</li>
	<li>Repeat steps 8.1.10-8.1.15 for about 10 neurons per coverslip/treatment.</li>
	<li>For sliced tissue, follow steps 8.18-8.22.</li>
	<li>Using the eye-port, search for the region of interest. For example, locate CA1 of the hippocampus.</li>
	<li>Turn off the eye-port and use the MAP2 channel to find the best plane of focus for the MAP2 signal with live scanning.</li>
	<li>Under the Acquire menu, choose Scan Large Image. Next, select the optical button&#39;s name with the previously configured acquisition settings under the Capturing panel from the panel that opens. Also, ensure to select the correct objective in this panel.</li>
	<li>Under the Area panel and the eyepiece, use the arrow keys to set the boundaries of the region of interest. Next, click on Save Large Image to File and create a save path filename for the image.</li>
	<li>Under the Setup panel, make sure Multichannel Capture is checked, and then choose the name of the optical button with the previously configured acquisition settings under Optical Conf. 
	NOTE: A z-stack for a large image is possible but will increase the scan time.</li>
</ol>
9. Analysis
<ol>
	<li>Similar to acquisition settings, use samples from all treatments to optimize threshold settings. Ensure that the threshold optimization focuses on decreasing background noise and enhancing the signal without oversaturating the intensity of the fluorescent signals. Once these settings are determined, apply the same threshold settings across all images used for analysis as described in steps 9.2-9.3.</li>
	<li>In ImageJ, the threshold option is located under the menu Image &#62; Adjust &#62; Threshold. Choose the Dark Background option if the image has a dark background.</li>
	<li>Next, adjust the upper and lower bounds of the threshold per previously determined optimized threshold settings, then click on Apply.</li>
	<li>For cultured cells, use the MAP2 channel and a freehand region of interest (ROI) tool to draw an ROI for each neuron, including dendrites and soma. For sliced tissue, draw a freehand ROI within the slice image that encapsulates the area of interest (e.g., stratum radiatum of the CA1 within the hippocampus).</li>
	<li>Obtain the area of the ROI. In ImageJ, measure the area under the menu Analyze &#62; Measure.</li>
	<li>Detect the number of puncta within each ROI using an automatic detection tool like Particle Analysis in ImageJ following steps 9.7-9.9.</li>
	<li>Find the Particle Analysis option under the menu Analyze &#62; Analyze Particles. First, define the puncta size diameter, typically 0.1-3 &#181;m2.</li>
	<li>Next, choose the Overlay Masks option from the Show drop-down menu and check the Display Results option. Then, click on OK.</li>
	<li>If puncta are not detected with the diameter range chosen, adjust the range until all puncta are detected with this analysis. Make sure to use the same Particle Analysis settings for all images.</li>
	<li>Divide the number of puncta by the area of an individual region of interest by following steps 9.11-9.13.</li>
	<li>Copy and paste the results for each image from the Results pop-up from ImageJ into a spreadsheet.</li>
	<li>First, identify which file and sample the data were obtained from. Then, divide the area of the ROI by the number of puncta.</li>
	<li>Then, clear the data from the&#160;Results&#160;pop-up, and repeat steps 9.2-9.12.</li>
	<li>Normalize results to control samples: Average the results (number of puncta/ROI area) for the control samples. Then, divide the obtained results of all samples by the average of the control to obtain the normalized results. The new average of the control samples should be equal to 1.</li>
</ol>

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The authors report no conflict of interest.

DetectSyn is a rapid assay that uses a proximity ligation assay to detect proteins within 40 nm of each other, which allows for the detection of synapse formation. This technique improves current fluorescent assays, which serve only as proxy measurements for synapse formation. DetectSyn detects quantifiable changes in synaptic proteins localized within 40 nm, i.e., within the synaptic cleft, of each other. Further, DetectSyn is more cost-effective and takes less time than techniques, like electron microscopy and array to...

Synapses are the fundamental unit of communication between neurons1. Many synapses between neurons within the same regions give rise to circuits that mediate behavior2. Synapses consist of a presynaptic terminal from one neuron that releases neurotransmitters or neuropeptides that relay information to postsynaptic receptors of another neuron. The summation of presynaptic signals determines whether the postsynaptic neuron will fire an action potential and propagate the message to other neurons.
Synaptopathology, the break down of synapses, arises in diseases and disorders marked by decreased neural volume, like Alzheimer&#39;s disease and major depressive disorder, resulting in circuits that no longer optimally perform3,4,5. Restoring synapse density likely underlies the efficacy of potential treatments for these disorders. For example, it was recently demonstrated that increasing synapses underlies the behavioral efficacy of rapid antidepressants6. To rapidly screen possible synaptopathology treatments, researchers require techniques that quickly identify changes to synapse numbers.
Current methodologies are either time-consuming and expensive (electron microscopy, array tomography), or they only examine postsynaptic changes without incorporating presynaptic engagement (spine analyses, immunofluorescence/colocalization). Dyes like DiI or fluorescent proteins like GFP help visualize neurons and characterize postsynaptic spines. However, spine analysis uses researcher-defined ratios to determine morphology, which can decrease reproducibility7. Further, how the different spine classes relate to functional synapses is still being uncovered8. Spine formation can be transient and may reflect postsynaptic plasticity, but these spines could be eliminated before stabilizing into a synapse with a presynaptic neuron9.
Colocalization provides a better proxy for synapses than spine analysis because one can immunostain for presynaptic and postsynaptic proteins. However, synaptic proteins may yield low colocalization values because the proteins are juxtaposed and may not consistently overlap. Thus, because the proteins are not wholly superimposed, colocalization techniques may not accurately measure changes to synapse formation due to this missing information. Finally, although both electron microscopy (EM) and array tomography provide high-resolution images of synapses, they are time-consuming. EM further requires specialized equipment, and researchers are limited to small volumes of tissue for any given experiment. While array tomography elegantly provides the ability to screen for many proteins on ultrathin sections and can be combined with EM10, this technique may be too labor-intensive and beyond the scope of experiments that need to scan rapidly for changes to synapse formation.
DetectSyn is a specific application of the Duolink Proximity Ligation Assay. The PLA assay allows for the general detection of protein-protein interactions. DetectSyn bridges proxy postsynaptic measures by amplifying a fluorescent signal emitted by tagged pre- and postsynaptic proteins within 40 nm of each other. If the synaptic proteins are within 40 nm, as within a synaptic cleft, then the secondary antibodies, which contain DNA probes, will hybridize into circular DNA. This hybridized circular DNA expresses a fluorescent probe, which is then amplified and detected with standard fluorescent microscopy techniques (see Figure 1). Crucially, unlike EM and array tomography, this technique does not require specialized equipment and takes about the same amount of time as standard immunohistochemistry. The accessibility of this technique, thus, enables investigators outside of research-intensive institutions to participate in synaptopathology research. Further, this technique can examine changes to synaptic density in multiple brain regions within a single experiment, offering a more holistic representation of synaptic changes due to disease or treatment.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10x PBS</td>
			<td>Fisher Scientific</td>
			<td>BP39920</td>
			<td>PBS made in house works, as well.</td>
		</tr>
		<tr>
			<td>24 well plates</td>
			<td>Fisher Scientific</td>
			<td>FB012929</td>
			<td>For tissue slices, pre-sterilized plates may be unnecessary.</td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminium foil</td>
			<td>Fisher Scientific</td>
			<td>15-078-290</td>
			<td></td>
		</tr>
		<tr>
			<td>Chicken anti-MAP2 antibody</td>
			<td>Abcam</td>
			<td>ab5392</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear nail polish</td>
			<td>Fisher Scientific</td>
			<td>NC1849418</td>
			<td>Other clear nail polish works, as well.</td>
		</tr>
		<tr>
			<td>Cold block</td>
			<td>Fisher Scientific</td>
			<td>13131012</td>
			<td></td>
		</tr>
		<tr>
			<td>Computer workstation</td>
			<td>HP</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal or fluorescent microscope</td>
			<td>Nikon</td>
			<td>A1R HD25</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-chicken FITC</td>
			<td>Fisher Scientific</td>
			<td>SA1-72000</td>
			<td></td>
		</tr>
		<tr>
			<td>Duolink donkey anti-Mouse PLUS</td>
			<td>Sigma</td>
			<td>DUO92001</td>
			<td></td>
		</tr>
		<tr>
			<td>Duolink donkey anti-Rabbit MINUS</td>
			<td>Sigma</td>
			<td>DUO92005</td>
			<td></td>
		</tr>
		<tr>
			<td>Duolink In Situ Detection Reagents Far Red</td>
			<td>Sigma</td>
			<td>DUO92013</td>
			<td>Contains ligation stock, amplification stock, ligase, and polymerase.</td>
		</tr>
		<tr>
			<td>Duolink In Situ Mounting Medium with DAPI</td>
			<td>Sigma</td>
			<td>DUO82040</td>
			<td></td>
		</tr>
		<tr>
			<td>Duolink In Situ Wash Buffers, Fluorescence</td>
			<td>Sigma</td>
			<td>DUO82049</td>
			<td>Contains Wash Buffer A and Wash Buffer B; dilute Wash Buffer B to 1% in diH20 for 1% Wash Buffer B.</td>
		</tr>
		<tr>
			<td>Fine-tipped paintbrush</td>
			<td>Fisher Scientific</td>
			<td>NC9691026</td>
			<td>Sable hair, size 00 or 000, can also find at craft stores</td>
		</tr>
		<tr>
			<td>Fisherbrand Cover Glasses: Rectangles</td>
			<td>Fisher Scientific</td>
			<td>12545MP</td>
			<td>Cover glass is unnecessary for cultured neurons already on glass coverslips.</td>
		</tr>
		<tr>
			<td>Fisherbrand Superfrost Plus Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>1255015</td>
			<td>For cultured neurons already on glass coverslips, Superfrost slides may be unnecessary.</td>
		</tr>
		<tr>
			<td>Freezer, -20&#176;C</td>
			<td>VWR</td>
			<td>76449-108</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips</td>
			<td>Fisher Scientific</td>
			<td>125480</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Fisher Scientific</td>
			<td>BP381-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Image processing software</td>
			<td>e.g. NIS Elements, ImageJ</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Fisher Scientific</td>
			<td>15-015-2633</td>
			<td></td>
		</tr>
		<tr>
			<td>Large petri dish, 100mm</td>
			<td>Fisher Scientific</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular grade water</td>
			<td>Fisher Scientific</td>
			<td>BP24701</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-Synapsin1 antibody</td>
			<td>Synaptic Systems</td>
			<td>106-011</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal donkey serum</td>
			<td>Jackson ImmunoResearch</td>
			<td>017-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>Fisher Scientific</td>
			<td>02-106-1013</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Fisher Scientific</td>
			<td>13-374-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips</td>
			<td>Fisher Scientific</td>
			<td>02-707-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes</td>
			<td>Fisher Scientific</td>
			<td>14-388-100</td>
			<td>Working volumes range from 3 &#181;L to 500 &#181;L</td>
		</tr>
		<tr>
			<td>Plastic pasteur pipette</td>
			<td>Fisher Scientific</td>
			<td>02-708-006</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision tweezers/foreceps</td>
			<td>Fisher Scientific</td>
			<td>12-000-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-PSD95 antibody</td>
			<td>Abcam</td>
			<td>ab18258</td>
			<td>Other antibody pairs may work, as well, with optimization.</td>
		</tr>
		<tr>
			<td>Refrigerator</td>
			<td>VWR</td>
			<td>76470-402</td>
			<td></td>
		</tr>
		<tr>
			<td>Small petri dish, 60 mm</td>
			<td>Fisher Scientific</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>Timer</td>
			<td>Fisher Scientific</td>
			<td>14-649-17</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Fisher Scientific</td>
			<td>BP337-100</td>
			<td></td>
		</tr>
	</tbody>
</table>

detectsyn: a rapid, unbiased fluorescent method to detect changes in synapse density

Synapses are the site of communication between neurons. Neuronal circuit strength is related to synaptic density, and the breakdown of synapses is characteristic of disease states like major depressive disorder (MDD) and Alzheimer's disease. Traditional techniques to investigate synapse numbers include genetic expression of fluorescent markers (e.g., green fluorescent protein (GFP)), dyes that fill a neuron (e.g., carbocyanine dye, DiI), and immunofluorescent detection of spine markers (e.g., postsynaptic density 95 (PSD95)). A major caveat to these proxy techniques is that they only identify postsynaptic changes. Yet, a synapse is a connection between a presynaptic terminal and a postsynaptic spine. The gold standard for measuring synapse formation/elimination requires time-consuming electron microscopy or array tomography techniques. These techniques require specialized training and costly equipment. Further, only a limited number of neurons can be assessed and are used to represent changes to an entire brain region. DetectSyn is a rapid fluorescent technique that identifies changes to synapse formation or elimination due to a disease state or drug activity. DetectSyn utilizes a rapid proximity ligation assay to detect juxtaposed pre- and postsynaptic proteins and standard fluorescent microscopy, a technique readily available to most laboratories. Fluorescent detection of the resulting puncta allows for quick and unbiased analysis of experiments. DetectSyn provides more representative results than electron microscopy because larger areas can be analyzed than a limited number of fluorescent neurons. Moreover, DetectSyn works for in vitro cultured neurons and fixed tissue slices. Finally, a method is provided to analyze the data collected from this technique. Overall, DetectSyn offers a procedure for detecting relative changes in synapse density across treatments or disease states and is more accessible than traditional techniques.

Data modified from Heaney et al.6 are presented to demonstrate an experiment where increased synapse formation is expected (please see6 for more information and a more in-depth discussion of the mechanism). Previously, it was demonstrated that rapid antidepressants require activation of the inhibitory metabotropic receptor, GABAB (gamma-aminobutyric acid subtype B), to be effective13. Further, previous data indicated that rapid antidepressants increa...

Watch this Scientific Journal Video about DetectSyn: A Rapid, Unbiased Fluorescent Method to Detect Changes in Synapse Density at JoVE.com

DetectSyn: A Rapid, Unbiased Fluorescent Method to Detect Changes in Synapse Density

DetectSyn is an unbiased, rapid fluorescent assay that measures changes in relative synapse (pre- and postsynaptic engagement) number across treatments or disease states. This technique utilizes a proximity ligation technique that can be used both in cultured neurons and fixed tissue.

Synapses are the site of communication between neurons. Neuronal circuit strength is related to synaptic density, and the breakdown of synapses is ...

detectsyn-rapid-unbiased-fluorescent-method-to-detect-changes-synapse

Research

JoVE Journal

Biochemistry

3.1K Views.  Wake Forest School of Medicine. DetectSyn is an unbiased, rapid fluorescent assay that measures changes in relative synapse (pre- and postsynaptic engagement) number across treatments or disease states. This technique utilizes a proximity ligation technique that can be used both in cultured neurons and fixed tissue.

Video: DetectSyn: A Rapid, Unbiased Fluorescent Method to Detect Changes in Synapse Density

A Rapid Laser Probing Method Facilitates the Non-invasive and Contact-free Determination of Leaf Thermal Properties

Plants can produce valuable substances such as secondary metabolites and recombinant proteins. The purification of the latter from plant biomass can be streamlined by heat treatment (blanching). A blanching apparatus can be designed more precisely if the thermal properties of the leaves are known in detail, i.e., the specific heat capacity and thermal conductivity. The measurement of these properties is time consuming and labor intensive, and usually requires invasive methods that contact the sample directly. This can reduce the product yield and may be incompatible with containment requirements, e.g., in the context of good manufacturing practice. To address these issues, a non-invasive, contact-free method was developed that determines the specific heat capacity and thermal conductivity of an intact plant leaf in about one minute. The method involves the application of a short laser pulse of defined length and intensity to a small area of the leaf sample, causing a temperature increase that is measured using a near infrared sensor. The temperature increase is combined with known leaf properties (thickness and density) to determine the specific heat capacity. The thermal conductivity is then calculated based on the profile of the subsequent temperature decline, taking thermal radiation and convective heat transfer into account. The associated calculations and critical aspects of sample handling are discussed.

A method was developed to determine the specific heat capacity and thermal conductivity of leaf tissue by non-invasive, contact-free near infrared laser probing, which requires less than 1 min per sample.

Plants can produce valuable substances such as secondary metabolites and recombinant proteins. The purification of the latter from plant biomass can be ...

Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different downstream patient-to-patient transmission potentials, and require very different patient treatments. Thus, recent Zika outbreaks make it urgent to develop tools that rapidly discriminate these viruses in patients and trapped mosquitoes, to select the correct patient treatment, and to understand and manage their epidemiology in real time.
Unfortunately, current diagnostic tests, including those receiving 2016 emergency use authorizations and fast-track status, detect viral RNA by reverse transcription polymerase chain reaction (RT-PCR), which requires instrumentation, trained users, and considerable sample preparation. Thus, they must be sent to &#34;approved&#34; reference laboratories, requiring time. Indeed, in August 2016, the Center for Disease Control (CDC) was asking pregnant women who had been bitten by a mosquito and developed a Zika-indicating rash to wait an unacceptable 2 to 4 weeks before learning whether they were infected. We very much need tests that can be done on site, with few resources, and by trained but not necessarily licensed personnel.
This video demonstrates an assay that meets these specifications, working with urine or serum (for patients) or crushed mosquito carcasses (for environmental surveillance), all without much sample preparation. Mosquito carcasses are captured on paper carrying quaternary ammonium groups (Q-paper) followed by ammonia treatment to manage biohazards. These are then directly, without RNA isolation, put into assay tubes containing freeze-dried reagents that need no chain of refrigeration. A modified form of reverse transcription loop-mediated isothermal amplification with target-specific fluorescently tagged displaceable probes produces readout, in 30 min, as a three-color fluorescence signal. This is visualized with a handheld, battery-powered device with an orange filter. Forward contamination is prevented with sealed tubes, and the use of thermolabile uracil DNA glycosylase (UDG) in the presence of dUTP in the amplification mixture.

Current multiplexed diagnostics to detect Zika, chikungunya, and dengue viruses require complex sample preparation and expensive instrumentation, and are difficult to use in low resource environments. We show a diagnostic that uses isothermal amplification with target-specific strand displaceable probes to detect and differentiate these viruses with high sensitivity and specificity.

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different ...

Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction

The dissociation constant describes the interaction between two partners in the binding equilibrium and is a measure of their affinity. It is a crucial parameter to compare different ligands, e.g., competitive inhibitors, protein isoforms and mutants, for their binding strength to a binding partner. Dissociation constants are determined by plotting concentrations of bound versus free ligand as binding curves. In contrast, titration curves, in which a signal that is proportional to the concentration of bound ligand is plotted against the total concentration of added ligand, are much easier to record. The signal can be detected spectroscopically and by enzyme-linked immunosorbent assay (ELISA). This is exemplified in a protocol for a titration ELISA that measures the binding of the snake venom-derived rhodocetin to its immobilized target domain of &#945;2&#946;1 integrin. Titration ELISAs are versatile and widely used. Any pair of interacting proteins can be used as immobilized receptor and soluble ligand, provided that both proteins are pure, and their concentrations are known. The difficulty so far has been to determine the dissociation constant from a titration curve. In this study, a mathematical function underlying titration curves is introduced. Without any error-prone graphical estimation of a saturation yield, this algorithm allows processing of the raw data (signal intensities at different concentrations of added ligand) directly by mathematical evaluation via non-linear regression. Thus, several titration curves can be recorded simultaneously and transformed into a set of characteristic parameters, among them the dissociation constant and the concentration of binding-active receptor, and they can be evaluated statistically. When combined with this algorithm, titration ELISAs gain the advantage of directly presenting the dissociation constant. Therefore, they may be used more efficiently in the future.

A detailed protocol to perform a titration ELISA is described. Moreover, a novel algorithm is presented to evaluate titration ELISAs and to obtain a dissociation constant of binding of a soluble ligand to a microtiter plate-immobilized receptor.

The dissociation constant describes the interaction between two partners in the binding equilibrium and is a measure of their affinity. It is a crucial ...

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

A Rat Methyl-Seq Platform to Identify Epigenetic Changes Associated with Stress Exposure

As genomes of a wider variety of animals become available, there is an increasing need for tools that can capture dynamic epigenetic changes in these animal models. The rat is one particular model animal where an epigenetic tool can complement many pharmacological and behavioral studies to provide insightful mechanistic information. To this end, we adapted the SureSelect Target Capture System (referred to as Methyl-Seq) for the rat, which can assess DNA methylation levels across the rat genome. The rat design targeted promoters, CpG islands, island shores, and GC-rich regions from all RefSeq genes. 
To implement the platform on a rat experiment, male Sprague Dawley rats were exposed to chronic variable stress for 3 weeks, after which blood samples were collected for genomic DNA extraction. Methyl-Seq libraries were constructed from the rat DNA samples by shearing, adapter ligation, target enrichment, bisulfite conversion, and multiplexing. Libraries were sequenced on a next-generation sequencing platform and the sequenced reads were analyzed to identify DMRs between DNA of stressed and unstressed rats. Top candidate DMRs were independently validated by bisulfite pyrosequencing to confirm the robustness of the platform.
Results demonstrate that the rat Methyl-Seq platform is a useful epigenetic tool that can capture methylation changes induced by exposure to stress.

Here, we describe the protocol and implementation of Methyl-Seq, an epigenomic platform, using a rat model to identify epigenetic changes associated with chronic stress exposure. Results demonstrate that the rat Methyl-Seq platform is capable of detecting methylation differences that arise from stress exposure in rats.

As genomes of a wider variety of animals become available, there is an increasing need for tools that can capture dynamic epigenetic changes in these ...

Fluorescent Visualization of Mango-tagged RNA in Polyacrylamide Gels via a Poststaining Method

Native and denaturing polyacrylamide gels are routinely used to characterize ribonucleoprotein (RNP) complex mobility and to measure RNA size, respectively. As many gel-imaging techniques use nonspecific stains or expensive fluorophore probes, sensitive, discriminating, and economical gel-imaging methodologies are highly desirable. RNA Mango core sequences are small (19&#8211;22 nt) sequence motifs that, when closed by an arbitrary RNA stem, can be simply and inexpensively appended to an RNA of interest. These Mango tags bind with high affinity and specificity to a thiazole-orange fluorophore ligand called TO1-Biotin, which becomes thousands of times more fluorescent upon binding. Here we show that Mango I, II, III, and IV can be used to specifically image RNA in gels with high sensitivity. As little as 62.5 fmol of RNA in native gels and 125 fmol of RNA in denaturing gels can be detected by soaking gels in an imaging buffer containing potassium and 20 nM TO1-Biotin for 30 min. We demonstrate the specificity of the Mango-tagged system by imaging a Mango-tagged 6S bacterial RNA in the context of a complex mixture of total bacterial RNA.

Here we present a sensitive, rapid, and discriminating post-gel staining method to image RNAs tagged with RNA Mango aptamers I, II, III, or IV, using either native or denaturing polyacrylamide gel electrophoresis (PAGE) gels. After running standard PAGE gels, Mango-tagged RNA can be easily stained with TO1-Biotin and then analyzed using commonly available fluorescence readers.

Native and denaturing polyacrylamide gels are routinely used to characterize ribonucleoprotein (RNP) complex mobility and to measure RNA size, ...

Rapid and Cost-Effective RNA Extraction of Rat Pancreatic Tissue

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective denaturation of proteins from RNA, 3) ribonuclease inactivation, and 4) removal of contamination from DNA, proteins, and carbohydrates. However, it is very laborious to maintain the integrity of RNA when there are high levels of RNase in the tissue. Spontaneous autolysis makes it very difficult to extract RNA from pancreatic tissue without damaging it. Thus, a practical RNA extraction method is needed to maintain the integrity of pancreatic tissues during the extraction process. An experimental and comparative study of existing protocols was carried out by obtaining 20-30 mg of rat pancreatic tissues in less than 2 minutes and extracting the RNA. The results were assessed by electrophoresis. The experiments were carried out three times for generalization of the results. Immersing pancreatic tissue in RNA stabilization reagent at -80 &#176;C for 24 h yielded high integrity RNA, when the RNA extraction reagent was used as the reagent. The results obtained were comparable to the results obtained from commercial kits with spin column bindings.

The purity and integrity of the isolated RNA is a vital step in RNA dependent assays. Here, we present a practical, rapid, and inexpensive method to extract RNA from a small quantity of undamaged pancreatic tissue.

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective ...

Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and isopycnic density gradient centrifugation. The major advantage of this procedure is that it does not rely on the sole use of solubilizing detergents to obtain subcellular fractions. This makes it possible to separate the plasma membrane from other membrane-bound organelles of the cell. This procedure will facilitate the determination of protein localization in cells with a reproducible, scalable, and selective method. This method has been successfully used to isolate cytosolic, nuclear, mitochondrial, and plasma membrane proteins from the human monocyte cell line, U937. Although optimized for this cell line, this procedure may serve as a suitable starting point for the subcellular fractionation of other cell lines. Potential pitfalls of the procedure and how to avoid them are discussed as are alterations that may need to be considered for other cell lines.

This fractionation protocol will allow researchers to isolate cytoplasmic, nuclear, mitochondrial, and membrane proteins from mammalian cells. The latter two subcellular fractions are further purified via isopycnic density gradient.

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and ...

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.
This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors &#8212; anthraquinones or anthraquinone-like molecules with one to three substituents &#8212; at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time.&#160;

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...