This protocol can be used to evaluate integrity and plasticity of a neuromuscular junction under normal and pathological conditions. This technique is a to simultaneously label the Schwann cells and pre and post lab animals on single muscle sections. After euthanizing the adult male rat, place the rat in the hood and open the thoracic cavity to access the heart using scissors and forceps.
Insert and intravenous catheter from the left cardiac ventricle toward the aorta and cut the right oracle. Next, start the perfusion with 100 milliliters of 0.9%normal saline until the blood exiting from the right oracle is clear. Then continue to perfuse with 250 to 300 milliliters of 4%paraformaldehyde in 0.1 molar phosphate buffer or PB pH 7.4 for 10 minutes.
After perfusion, remove the skin of the bilateral hind limb using a surgical blade and expose the bilateral sciatic nerve and medial gastrocnemius muscle. Dissect out the bilateral medial gastrocnemius muscle carefully from the hind limb using a blade and postfix the entire muscle in 10 milliliters of 4%paraformaldehyde. After two hours, remove the muscle from the solution and cryoprotect it in 10 milliliters of 25%sucrose in 0.1 molar PB for one day at four degrees celsius.
Once the muscular tissue is immersed in the sucrose solution, fix it on a freezing stage of a sliding microtome system using a frozen section medium. Slice the muscle horizontally along the long axis of the muscle at the thickness of 80 micron. Using a brush, place seven cryo sections per well in an orderly fashion in a six well culture plate with 10 milliliters of 0.1 molar PB.For fluorescent staining, incubate four sections per well with 1.5 milliliters of 0.1 molar PB containing 75 microliters of 10%Triton X 100 and 45 microliters of normal donkey serum on an orbital shaker at 20 rpm.
After 30 minutes, transfer the sections into 1.5 milliliters of a mixed solution of primary antibodies containing rabbit anti-neuro filament 200, goat anti-vesicular acetylcholine transporter in 0.1 molar PB, in 1%normal donkey serum, and 0.5%Triton X 100 and incubate overnight at four degrees Celsius. The next day, wash the sections three times for five minutes each in 0.1 molar PB on in orbital shaker at 20 rpm. After the last wash, incubate the section in a mixed solution of secondary antibodies containing donkey anti-rabbit AF 488 and donkey anti-goat AF 546, as well as the biomarkers of alpha bungaro toxin AF 647 and phalloidin 350 containing in 0.1 M PB containing 1%normal donkey serum and 0.5 Triton x 100 for one hour, Protecting them from light.
After incubation, wash the sections in PB and mount them on a microscope slide. Apply cover glass to the section using 50%glycerin in distilled water. Observe and image the specimen using a con focal imaging system equipped with a 20 x subjective lens with a numerical aperture of 0.75 and a 40 x lens with a numerical aperture of 0.95.
For multiple fluorescent imaging, set the excitation and emission wavelengths of each biomarker as described in the text manuscript. Set the resolution of image capture to 640 by 640 pixels. Next, set the start and end focal plane.
Set step size to either one or two microns. Then choose depth pattern, image capture, and Z series. For the lower magnification images at 20 X, capture 40 Z stack images in two micron step size using a 105 micron pinhole.
Choose the projection or topography mode and intensity projection over Z axis to integrate all images in a single in focus. For the higher magnification images at 40 X, capture 80 Z stack images in one micron step size with zoom set to two and a 105 micron pinhole. Integrate all images in a single in focus.
Reconstruct the images in the three dimensional pattern with the image processing system. The representative spacial correlation of the medial gastrocnemius muscle with neuro filament 200 positive nerve fibers, vesicular acetylcholine transporter positive pre-synaptic terminal, alpha bungaro toxin positive post-synaptic acetylcholine receptors, S100 positive perisynaptic Schwann cells, phalloidin positive muscular fibers, and DAPI labeled cellular nuclei are demonstrated here. The neurofilament 200 positive nerve fibers ran in bundle.
These nerve fibers gave branches to the vesicular acetylcholine transporter positive presynaptic terminals, which formed a mirror relationship with the alpha bungaro toxin positive post-synaptic acetylcholine receptors. In addition, the S100 positive perisynaptic Schwann cells were detected around the neurofilament 200 positive nerve fibers close to the pre-synaptic terminals. The spatial correlation was further exhibited in a three dimensional pattern showing the detailed morphological characteristics of the neuromuscular junction from different perspectives.
In order to observe more imaging in muscular sections, it is critical to slice the muscle horizontally along the long axis at a thickness of 80 micron.
