Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

The overall goal of the following experiment is to delineate single schwan cells at the neuromuscular junction using confocal microscopy. This protocol uses transgenic mice that express GFP in terminal and axonal schwan cells. The first step is to dissect out a nerve muscle explan.

Next sequential bleaching of all but one schwan cell at a given neuromuscular junction is performed revealing single cell morphology. Finally, a low power map is created. The tissue is fixed and used for immunohistochemistry to visualize any desired marker in relation to the remaining unbleached schwan cell.

The results show single cell morphology and if desired time-lapse microscopy or immunohistochemical colocalization analysis in fixed tissue based on confocal imaging. Though this method can provide insight into the morphology and territory of single swan cells at the neuromuscular junction. It can also be applied to other systems such as cell lines, living brain slices, or even other species like zebrafish.

Generally, individuals new to this method will struggle because it takes some experience to identify a sufficiently superficial neuromuscular junction and not waste time on structures that are just not worth the effort Prior to starting the surgery, oxygenate and cool the ringer solution for at least 15 minutes. Place a 15 centimeter tissue culture dish with ice under a microscope. Cover it with a metal plate.

Place the 10 centimeter tissue culture dish filled with ringer solution on the metal plate. Place the oxygenation stone into solution. The next step is to select a mouse that has its swan cells and axons labeled in different colors.

After the mouse has been euthanized, spray it with ethanol To prevent fur contamination, use large scissors to make a midline incision over the sternum and two incisions parallel to the lower edge of the rib cage. Remove the skin, open the abdomen and dissect off the pectoral muscles with incisions close to the muscle insertion at the sternum. Use forceps to hold onto the xiphoid cartilage and cut the diaphragm open just below the xiphoid cartilage.

Dissect out the diaphragm along its costal insertions. Occasionally wet the tissue with ringer solution to prevent drying using forceps to hold the rib cage by the xiphoid process. Start cutting the ribs off the vertebral column as close as possible to their insertions.

The left and right cuts should converge above the heart towards the manubrium stern eye. Try to avoid cutting major blood vessels, especially the subclavian veins. For better visibility.

Gently lift the preparation while holding onto the xiphoid process with forceps. Now make a transverse cut just below the manubrium stern eye to remove the thorax and transfer the X explan into the 10 centimeter dish with the prepared ringer solution. Place the dish on the metal plate and ice filled dish under the microscope under the microscope.

Using small spring scissors, remove the remnants of thymus, pleura, diaphragm, and pectoral muscles. Using small spring scissors, dissect off all the ribs that do not insert at the sternum. Cut the tissue between the ribs.

This allows the X explan to fit into a 3.5 centimeter SIL guard coated dish. Now using eight to 10 shortened minuchin pins, pin down the X explan, avoiding the triangular stern eye muscle pointed out here. Two pins should go through the cartilaginous parts of the sternum, and two pins should go through the softer cartilaginous parts between the ribs.

Avoid puncturing the ribs under or close to the triangulars muscle. Continue pinning until the ribs are somewhat spread. Fixing the muscle into a slightly stretched position.

Transfer the 3.5 centimeter sill guard coated dish to a confocal microscope equipped with an Argonne laser and water immersion objectives. For a time-lapse microscopy, insert the 3.5 centimeter sill guard coated dish into the heating ring. Install a superfusion system and install a temperature probe.

Make sure that nothing touches the explan, the rim of the dish, or the cigar coating. For time-lapse microscopy, heat the X explan, but for schwan cell bleaching room temperature is better. Now with a Forex air objective, observe the innervation pattern of the triangular stern eye muscle and find the triangular stern eye end plate band.

Switch to the 20 x dipping cone objective and start looking for superficial regions within the nplate band areas. Overlying the ribs and close to the intercostal nerves are good candidate regions. Switch to the 100 x or 60 x dipping cone Objective to check on individual and mjs.

Ideally locate an NMJ covered by several schwan cells that is located very superficially Critical for the success of this protocol is to find their superficial and flat neuromuscular junction. Since the neuromuscular junction is innovating cylindrical muscle fibers, it tends to be wrapped around the fiber and hands often does not have the perfect angle for imaging. Select only the neuromuscular junctions that are superficial flat and on the surface Now using a 100 x 1.0 numerical aperture objective.

With a 2.5 zoom, acquire a confocal image of the NMJ. These settings provide a pixel size of about 0.1 micrometers, ensuring proper spatial sampling according to the nyquist criterion. Now photobleach the schwan cell by parking the 488 nanometer laser beam centrally on the nucleus using maximal power for five seconds.

Next, refocus on the cell and judge the bleaching result. If necessary, repeat the bleaching step again until the GFP levels are reduced to near background levels, then acquire another image. It is critical to make sure that the bleached region is outside of any overlap between two cells.

If there is overlap, bleaching in a second cell will be obvious. When done properly, this approach results in a clearly revealed final cells, often with a quite surprising morphology that one could not have guessed before starting the procedure. Repeat the bleaching process until all but one of the schwan cells are bleached.

The last unbleached schwan cell is suitable for confocal time-lapse microscopy. After completing the time-lapse imaging, make a map of these njs by taking an image at 100 x without zoom, then images of the region using the 20 x objective and repeat the process using the four x objective. This map is needed to find njs following immunohistic chemistry of a fixed muscle.

After imaging and bleaching unpin the explan and transfer it to a 50 milliliter tube with at least 15 milliliters of 4%PFA. Then place the tube on ice for an hour after chilling the tissue for an hour, rinse it with 0.1 molar glycine in PBS for at least 10 minutes. Then transfer the fixed explan to a five centimeter silk guard coated tissue culture dish filled with 0.01 molar PBS and prepare to further dissect the explan.

First, cut out a trapezoid shape with one side parallel to the sternum and the other through the bone to cartilage transitions. This contains the triangulars muscle on its surface. Next, remove the lower ribs with a horizontal or transsternal cut so that the coddle end of the triangulars muscle can be freely accessed.

Now insert a hypodermic needle as a pin into the upper part of the trapezoid. Next, remove the triangulars muscle. First, use forceps to gently hold the coddle corner of the muscle.

And second, use a syringe and needle like a micro scalpel to cut parallel to the plane of the thoracic wall. Once the coddle part of the muscle is released, insert another needle below the muscle through the thoracic wall to immobilize the preparation. While cutting the connective tissue insertions, peel away the muscle from the thorax with gentle pulling from the forceps.

Finally, remove fat and blood vessel remnants using forceps. Be careful not to rip away the nerves. The triangulars muscle is now ready for immunohistochemistry.

A triangulars stern eye explan using mice expressing GFP under the PLP promoter was prepared for ex vivo visualization of schwan cells. Axons could be simultaneously visualized by crossing P-L-P-G-F-P to thigh one OFP three mice. Using the described protocol, the morphology of single terminal schwan cells was revealed in surprisingly high detail by bleaching the neighboring two terminal and the first axonal schwan cells live.

Imaged neuromuscular junctions were tracked back using the map created beforehand and scanned at high resolution in the fixed tissue following live confocal microscopy, the triangular stern explan was fixed and stained. Shown here are acetylcholine receptors labeled with bungalow toin coupled to Alexa 6 47 in red and synaptic vesicles labeled with an antis synaptophysin antibody in white. After watching this video, you should have a good understanding of how to visualize the morphology of single cells, which were initially all labeled by the same fluorescent protein and packed too densely to resolve individually.

Our example shows the neuromuscular junction, but there are many other potential applications.