DetectSyn takes advantage of the proximity ligation assay technology to quickly identify changes to synapses. We also provide a protocol to analyze the results. The main advantage of DetectSyn is how quickly the results can be obtained in the large regions of tissue that can be analyzed.
To begin, carefully remove the blocking solution with a plastic Pasteur pipette without disturbing the cells. Then line a large plastic Petri dish with Parafilm. And using forceps, carefully transfer the cover slips to the Parafilm.
Add 60 microliters of the primary antibody solution to the top of the cover slip, ensuring that the antibody solution does not spill over the sides of the cover slip. To provide humidity and prevent samples from drying out during incubation, add ultrapure water to a smaller Petri dish, and carefully place the dish around the cover slips. Then cover the larger Petri dish and incubate the cultured cells for one hour at room temperature.
After incubation with the primary antibody, use forceps to carefully tap off the primary antibody solution from the cover slips onto a paper towel and transfer the cover slips to their original 24-well plate filled with 500 microliters of PBS. Then wash the samples with PBS three times for 10 minutes each with gentle agitation on an orbital shaker. After the last wash, transfer the cover slips back to the large Petri dish and add 40 microliters of the secondary antibody solution to the top of the cover slip.
Cover the Petri dish and incubate the samples at 37 degrees Celsius for one hour. For ligation, while keeping the ligase on a cold block, dilute the ligase 1 to 40 using the ligation stock and immediately add 40 microliters of the ligase mix to the cover slips. Cover the Petri dish and incubate the samples at 37 degrees Celsius for 30 minutes.
For amplification, dilute the polymerase 1280 on a cold block using the amplification stock. Then add 40 microliters of the amplification mix to the cover slips and incubate the samples for 100 minutes at 37 degrees Celsius. Next, for mounting, drop three microliters of mounting media onto a slide.
Tap off excess wash buffer from the cover slip and place the cover slip with cells facing down into the mounting media. Seal the sides with a small amount of clear nail polish to hold the cover slip in place. For mounting of sliced tissue, carefully transfer a tissue slice to the prepared slide and lay it flat.
Then drop 5 to 10 microliters of mounting media onto the tissue slice. Place a glass cover slip over the tissue slice and seal the cover slip with clear nail polish, as demonstrated earlier. For digital imaging with a confocal microscope, adjust the gain, offset and laser power for each fluorescent channel to decrease background noise and enhance the fluorescent signal.
Ensure that the fluorescent signal is not oversaturated. For the culture neurons, under the ND Acquisition tab, click on Save to File. Then under Browse, choose a file to save the image and input the file name.
Next, under the Z tab, select the Symmetric Mode Defined by Range option. Set the focus to the best map two-plane and click on the Relative button to set this focal plane as the middle of the z-stack. Next, set the range to five micrometers with one micrometer steps and 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 Configuration and click on Run Now. For sliced tissue, from the acquire menu choose Scan Large Image, then under the capturing panel, select the optical button with the previously configured acquisition settings. Also, select the correct objective in this panel.
Next, under the area panel and eye piece, use the arrow keys to set the boundaries of the region of interest or ROI. Then click on Save large image to file and create a save path file name for the image. Finally, under the setup panel, ensure that Multichannel Capture is checked.
Then choose the name of the optical button with the previously configured acquisition settings under optical configuration. In ImageJ, open the threshold option by going to the image menu, clicking on Adjust, and then Threshold. Choose the Dark Background option if the image has a dark background.
Next, adjust the upper and lower bounds of the threshold per previously determined optimized threshold settings and click on Apply. For cultured cells, use the MAP2 channel and a free hand ROI tool to draw an ROI for each neuron, including dendrites and soma. For sliced tissue, draw a free hand ROI within the slice image that encapsulates the area of interest.
Then obtain the area of the ROI by opening the Analyze menu and clicking on Measure. To detect the number of punta within each ROI, use an automatic detection tool like particle analysis. From the Analyzed menu, select Analyzed Particles and define the puncta size diameter.
Next, from the show drop-down menu, choose the Overlay Masks option, then check the Display results option and click on Okay. To divide the number of punta by the area of the individual ROI, copy and paste the results for each image from the results pop up into a spreadsheet. Identify which file and sample the data we're obtained from, and divide the area of the ROI by the number of punta.
Finally, to normalize the results to control samples, average the results for the control samples and divide the obtained results of all samples by the average of the control. The GABAB agonist, baclofen, is required for the in vitro efficacy of the rapid antidepressant Ro-256981. An increase in synapse formation is demonstrated by an increase in white punta.
Without baclofen, no increase in synapse formation is seen. In vivo, intraperitoneal injection of the rapid antidepressant results in an increase in synapse formation compared to the saline control. While some punta appear in the technical control images, they are generally not the same size as demonstrated by the white spec compared to the large punta in the other panels.
Further, these punta are not in the same location as demonstrated by some punta appearing within the soma. DetectSyn can identify changes to synapses due to a disease state or drug treatment. This method can help provide insight to any area of research that studies the disorders affecting the development or breakdown of synapses.
