Visualizing the Morphological Characteristics of Neuromuscular Junction in Rat Medial Gastrocnemius Muscle

This study was approved by the Ethics Committee of Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences (approval No. 2021-04-15-1). All procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington, D.C., 1996). Three adult male rats (Sprague-Dawley, weight 230 ± 15 g) were used. The rats were housed in a 12 h light/dark cycle with controlled temperature and humidity and with free access to food and water. The instruments and materials for this study are shown in Figure 1.

1. Perfusion

1. Inject 250 mg/kg tribromoethanol solution intraperitoneally to induce euthanasia of the rats.
2. Once breathing stops, open the thoracic cavity to access the heart using scissors and forceps. Insert an intravenous catheter from the left cardiac ventricle toward the aorta, and then cut the right auricle.
3. Perfuse the experimental rats in the hood. Start by perfusing with 100 mL of 0.9% normal saline until the blood exiting from the right auricle is clear, then continue to perfuse with 250-300 mL of 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) for about 10 min until the tail is hard to bend.

2. Regional anatomy

1. After perfusion, remove the skin of the bilateral hind limb using a surgical blade (Figure 2A) and expose the bilateral sciatic nerve and medial gastrocnemius muscle (Figure 2B).
2. Dissect out the bilateral medial gastrocnemius muscle carefully from the hind limb using a blade (Figure 2C) and post-fix the entire muscle in 10 mL of 4% paraformaldehyde in 0.1 M PB for 2 h.
3. Remove the muscle from the solution and cryoprotect the muscle in 10 mL of 25% sucrose in 0.1 M PB for 1 day at 4 °C until the muscle is completely immersed in the solution.

3. Cryosectioning of the muscle

1. Once the muscular tissue is immersed in the solution, fix it on a freezing stage of a sliding microtome system using frozen section medium. Slice the muscle horizontally along the long axis of the muscle at the thickness of 80 µm.
2. Place seven cryo-sections per well in an orderly fashion in a six-well culture plate with 10 mL of 0.1 M PB (pH 7.4) using a brush.
   NOTE: The sections are placed in order so that the sections in different wells are adjacent, which makes the sections used for different stains more consistent.

4. Multiple fluorescent staining with NF200, VAChT, α-BTX, and Pha

NOTE: Multiple fluorescent staining with NF200, VAChT, α-BTX, and Pha was applied to reveal the nerve fibers, pre-synaptic terminals, post-synaptic nicotinic acetylcholine receptors, and muscular fibers, respectively.

1. Incubate four sections per well with 1.5 mL of 0.1 M PB containing 75 μL of 10% Triton X-100 (0.5%) and 45 μL of normal donkey serum (3%) on an orbital shaker at 20 rpm for 30 min.
2. Transfer the sections into 1.5 mL of mixed solution of primary antibodies containing rabbit anti-NF200 (1:1,000), goat anti-VAChT (1:500) in 0.1 M PB (pH 7.4) with 1% normal donkey serum, and 0.5% Triton X-100, and incubate overnight at 4 °C. The next day, wash the sections 3x for 5 min each in 0.1 M PB (pH 7.4) on an orbital shaker at 20 rpm.
3. Incubate the sections in 1.5 mL of mixed solution of secondary antibodies containing donkey anti-rabbit AF488 (1:500) and donkey anti-goat AF546 (1:500), as well as the biomarkers of α-BTX, AF647 (1:500), and phalloidin 350 (1:500) in 0.1 M PB (pH 7.4) containing 1% normal donkey serum and 0.5% Triton X-100 for 1 h at room temperature. Protect from light.
4. After washing 3x for 5 min each in 0.1 M PB (pH 7.4), mount the sections on microscope slides. Apply cover glass to the sections using 50% glycerin in distilled water for observation.

5. Multiple fluorescent staining with NF200, S100, α-BTX, and DAPI

NOTE: The procedures are similar to that of step 4; the main difference is between the primary antibodies and their corresponding secondary antibodies.

1. Use the following primary antibodies: chicken anti-NF200 (1:6,000), rabbit anti-S100 (1:2,500) in step 4.2 and their corresponding secondary antibodies: donkey anti-chicken AF488 (1:500) and donkey anti-rabbit AF546 (1:500), as well as the biomarkers of α-BTX AF647 (1:500) and DAPI (1:50000) in step 4.3.

6. Observation and analyses

1. Observe the specimen and take images using a confocal imaging system equipped with the following objective lenses: 20x lens with NA of 0.75 and 40x lens with NA of 0.95.
2. Use excitation and emission wavelengths of 350 nm (Pha), 488 nm (NF200), 546 nm (VAChT and S100), 647 nm (α-BTX), or DAPI corresponding to the multiple fluorescent staining. Set the resolution of image capture to 640 x 640 pixels.
   NOTE: The 640 x 640 pixels resolution is chosen because the images were captured in Z-stacks. A 1024 x 1024 pixel resolution results in slow imaging and photobleaching in Z-stacks.
3. Set start focal plane and end focal plane. Set step size to either 1 µm or 2 µm. Choose depth pattern, image capture, and Z series.
4. For the lower magnification images taken at 20x, capture 40 images (Z-stacks) in 2 µm step size from each 80 µm thick section using a 105 µm pinhole. Choose the projection/topography mode and intensity projection over Z axis to integrate all images in a single in-focus.
5. For the higher magnification images taken at 40x, capture 80 images (Z-stacks) in 1 µm step size from each 80 µm thick section with zoom set to 2 and a 105 µm pinhole. Integrate all images in a single in-focus.
6. Reconstruct the images in the three-dimensional pattern with the image processing system as previously described in²⁷.