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

1. Triangularis Sterni Explant (Figure 1)

Timing: 15 min.

1. Prepare surgical instruments: 2 pairs of forceps, 1 pair of scissors, 1 pair of spring scissors, 1 tissue culture dish (10-cm). Pre-bubbled (minimum for 15 min) cooled (4 °C) Ringer’s solution with 5% CO₂/ 95% O₂. Fill 15-cm tissue culture dish with ice and cover it with metal plate. Prepare dissection microscope and place the 15-cm dish with ice under the dissection scope in order to cool the explant during dissection.
2. Lethally anesthetize the mouse by isoflurane overdose (or any other approved method of sacrifice). Spray the mouse with 70% ethanol to prevent fur contamination of the explant. For SC bleaching, we used Plp-GFP mice, which can be crossed to Thy1-OFP3 or Thy1-Membow13 for simultaneous visualization of the axon.
3. Using the pair of large scissors, make a midline incision of the skin over the sternum and two incisions parallel to the lower edge of the rib cage.
4. Using the pair of large scissors, remove the skin, open the abdominal wall, and make incisions parallel to the rib cage all the way back to the vertebral column. Then dissect off the pectoral muscles by making incisions close to the muscle insertion at the sternum.
5. Cut the diaphragm open just below the xiphoid cartilage and dissect off the diaphragm along its costal insertions.
6. Holding the rib cage by the xiphoid process with a set of forceps, 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 sterni. Try to avoid cutting major blood vessels (especially the subclavian veins). Better visibility is achieved by gently lifting the preparation while holding onto the xiphoid process with forceps.
7. Make a transverse cut just below the manubrium sterni and transfer the explant into the 10-cm dish with cooled 5% CO₂/ 95% O₂-bubbled Ringer’s solution. Place the 10-cm dish on the metal plate covering the 15-cm tissue culture dish filled with ice. All further steps should be done under the dissection microscope.
8. Using small spring scissors remove the remnants of thymus, pleura, diaphragm (inside) and pectoral muscles (outside; Figure 1B-D).
9. To fit the explant to the 3.5-cm dish, dissect off all the ribs that do not insert to the sternum. This is best done using small spring scissors and cutting the tissue in between ribs (Figure 1E).
10. Pin the triangularis sterni nerve-muscle explant into the Sylgard coated 3.5-cm tissue culture dish using minutien pins (shortened to < 4 mm; Figure 1G). Two pins should go through cartilaginous (white) parts of the sternum. Insert at least two pins through the ribs both on the left and right side of the explant aiming for the softer cartilaginous parts and avoiding the ribs under or close to the triangularis muscle. The more pins are used, the better (we typically use 8-10). As you pin down the preparation, make sure that the ribs are somewhat spread, fixing the muscle into a slightly stretched position.
11. Optional: Visualize synaptic sites containing acetylcholine receptors by adding bungarotoxin coupled to Alexa dyes (concentration of 0.8 μg/ml in 5% CO₂/ 95% O₂-bubbled Ringer’s solution). Incubate pinned explant in Sylgard coated dish for 15-20 min at room temperature then wash several times with 5% CO₂/ 95% O₂-bubbled Ringer’s solution.

2. Bleaching SCs and Optional Time-lapse Microscopy (Figure 2)

Timing: 30 – 45 min + optional 1 – 5 hr for time lapse.

1. Transfer 3.5-cm Sylgard-coated dish to a confocal microscope equipped with an argon laser (488 nm wavelength) and water immersion objectives (4x, 0.13 NA; 20x, 0.5 NA and 100x, 1.0 NA or 60x, 0.9 NA).
2. Optional for time-lapse microscopy: Insert 3.5-cm Sylgard-coated dish into heating ring, install superfusion system and temperature probe (Figure 2A). Make sure that none of them touch the explant, the rim of the dish or the Sylgard coating. Heat the sample to 33-35 °C. For SC bleaching without time-lapse, room temperature is better, as cells show less dynamics between bleaching steps.
3. With a 4x air objective observe innervation pattern of triangularis sterni muscle and find triangularis sterni endplate band. Switch to 20x dipping-cone objective and start looking for superficial regions within the endplate band (areas overlying ribs are good candidates). Change objective to 100x or 60x dipping-cone objective to check on individual NMJs. Ideal is an NMJ that is covered by several SCs and is located very superficially (Figure 2B). If you perform time-lapse imaging after bleaching, select up to 3 NMJs that are relatively close together to increase yield.
4. Acquire a confocal image of the NMJ before photo-bleaching individual SCs (Figure 2B). (For full resolution imaging, e.g. on an Olympus FV1000 microscope, use a 100x, 1.0 NA objective and 2.5 zoom, which results in Nyquist-limited sampling of ~0.1 μm per pixel.)
5. “Park” the 488 nm laser beam centrally on the nucleus of a SC using maximal power for 5 seconds (alternatively an efficient scan pattern in a small region of interest can be used, such as the “tornado scan” function on an Olympus microscope; as we use a confocal microscope, the laser is focussed into the image-conjugate planes and hence, at 488 nm with an objective of 1.0 N.A., < 0.5 μm in diameter). Re-focus on the cell and judge bleaching result. If necessary repeat bleaching step again until GFP levels are reduced to near-background levels. Acquire an image after bleaching (Figure 2C). 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.
6. Repeat step above until all but one SC are bleached (Figure 2D-E). The last “unbleached” SC is suitable for confocal time-lapse microscopy. Reconstruct single cells by subtraction of images, e.g. using Photoshop software, to delineate single SC morphology and territory (Figure 2F). Be aware that subtracting two mages adds noise (e.g. by importing them in consecutive “layers” in Photoshop and then setting the top channel to “difference” in the drop down menu at the top of the channels tab). This can be circumvented by using the “despeckle” function on the channels before subtraction and cropping away any background that obviously does not originate from the bleached cell. To optimize contrast, it can be necessary to adjust the brightness of the two channels in the “levels” window. The resulting image of the bleached cell is the superimposed on the original image of a synapse, pseudo-colored in a unique hue and made transparent to color the territory of the bleached cell in the original image (from doing this for all bleached and the last, unbleached cells the image shown in Figure 2F results).
7. Optional: Start confocal time-lapse microscopy after bleaching all but one SCs. Take an image at fixed intervals (for example every 5 to 10 min). Use as little laser power as possible. Up to 3 NMJs can be imaged at the same session.
8. Make a map of the time-lapsed NMJs by taking an image at 100x without zoom, then images of the region using 20x and 4x objectives (Figure 2G-I). This “map” is needed to find NMJs following immunohistochemistry of a fixed muscle.
9. For image processing, convert individual images into maximum intensity projections and save in tiff format. Combine all z-projected time-points into a stack and align in xy, e.g. using the “stackreg” plugin¹⁰ in Fiji (a package based on the open source software ImageJ; National Institutes of Health).

3. Fixation and Immunohistochemistry (Figure 3)

Timing: Overnight.

1. Transfer the explant into a 50-ml reaction tube containing at least 15 ml 4% PFA by carefully removing the pins. Post-fix the anterior thoracic wall containing the triangularis sterni muscle for 1 hr on ice. Rinse fixed tissue with 0.1 M glycine in PBS (to quench residual fixative and hence reduce background) for at least 10 min. Samples can be stored at this point and processed later.
2. Transfer fixed explant to a 10-cm Sylgard coated tissue culture dish filled with 0.01 M PBS. Cut out a trapezoid shape with one side parallel to the sternum and the other through the bone-to-cartilage transitions – this contains the triangularis muscle on its surface. Remove lower ribs with a horizontal (i.e. trans-sternal) cut so that the caudal end of the triangularis muscle can be freely accessed (Figure 3A-D).
3. Insert a hypodermic needle as a pin into the upper part of the trapezoid (Figure 3E). Use a hypodermic needle attached to a 1 ml syringe and forceps to remove the triangularis muscle on the surface by gently holding onto a caudal corner of the muscle and using the needle as a micro-scalpel. Make sure that cuts are made parallel to the plane of the thoracic wall. Once the caudal part of the muscle is released, insert another hypodermic needle as a pin below the muscle through the thoracic wall – this immobilizes the preparation and allows exerting a gentle pull on the muscle using forceps (Figure 3F). As you are cutting connective tissue insertions, “peel” away the muscle from the thorax. Cut last caudal attachment with spring scissors. Remove fat and blood vessel remnants using forceps. Be careful not to rip away the nerves. Note: Use separate sets of tools and dishes for working with live and fixed tissue.
4. Start immunohistochemistry (using a standard protocol) by transferring tissue samples into blocking solution for 1 hr on a shaker at room temperature. Smallest possible volume for staining is 200 μl using a 96-well plate.
5. Apply primary antibody diluted in blocking solution, 200 μl per well, overnight at 4 °C on a shaker.
6. Wash in 0.01 M PBS three times for 10 min.
7. Apply suitable secondary antibody for 1-2 hr at room temperature diluted in blocking solution.
8. Wash in 0.01 M PBS three times for 10 min.
9. Mount in anti-fading medium (e.g. Vectashield) and coverslip.
10. Place slide on magnetic metal plate, place magnet on top of the sample to flatten for a few hours or overnight at 4 °C. Seal with nail polish.