Neuroscience
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Toluidine Blue Staining of Resin-Embedded Sections for Evaluation of Peripheral Nerve Morphology
Chapters
Summary July 3rd, 2018
Here we present a protocol to visualize fine structures of peripheral nerves by obtaining and staining 1-2 µm sections with toluidine blue
Transcript
This method could help answer key questions in the fields of peripheral nerve injury and regeneration such as what is the morphometry of a peripheral nerve, how many axons are present, what is the state of myelination, and is there fibrotic tissue present? The main advantage of this technique is that it allows for high-resolution visualization of nerve features. Begin by placing an anesthetized rat in the prone position on the top of a dissection mat.
Place a nose cone to maintain anesthesia with 1-2%isoflurane. To ensure adequate depth of anesthesia test the animals for pedal withdrawal and palpebral reflexes of the hind feet. Once an appropriate depth of anesthesia is achieved, shave the hair of the hind limbs and sterilize the shaved areas with 70%ethanol.
Palpate the femur to identify the proper location for the incision. The femur is located just proximal to the most accessible segment of the sciatic nerve. After making a two to three centimeter incision in the skin along the hind limb from the knee up to the greater trochanter locate the plane between the biceps femoris muscle and the gluteus maximus muscle.
Then use microdissection scissors to separate the underlying fascia and expose two to three centimeters of the underlying sciatic nerve. Use a retractor to widen the gap between the two muscles. With fine forceps and iris scissors carefully separate the nerve from the surrounding connective tissue, taking great care not to compress or cut the nerve.
Next, cover the exposed nerve with Trump's Fixative and let sit for 10 minutes. If necessary, place gauze under the hind leg to improve the angle so that more fixative can be added into the cavity. Remove the fixative after 10 minutes and repeat this step two more times.
Under the dissecting microscope cut the sciatic nerve from both sides using fine dissection scissors, making sure not to stretch or pinch the nerve. Immediately put the nerve sections in 15 mL tubes containing Trump's Fixative and fix at four degrees Celsius for one week, changing the fixative every 48 hours. Post-fixation, carefully remove any remaining fat and connective tissues from the nerve, and use a sharp scalpel to cut the nerve into segments approximately five millimeters in length.
Nerve segments can be separated into different labeled tubes. Immerse nerve segments in freshly prepared 2%osmium tetroxide for two hours. Using an epoxy embedding kit, prepare the final embedding mixture.
Mix the epoxy embedding medium with DDSA solution and mix the epoxy embedding medium with NMA solution. Mix both solutions for at least 20 minutes with a magnetic stirrer. Immediately before use, combine the accelerator DPM to solution B and then combine this with solution A such that the accelerator's final proportion is between one point five and 2%of the total volume.
Transfer the nerve segments to one point five milliliter tubes and after washing with PBS, start the dehydration process by the replacing the PBS with increasing concentrations of acetone and distilled water for 10 minutes each then in 100%acetone three times for 10 minutes each. One critical step is the tissue dehydration. Water and resin will not interact, so any water remaining in the tissue will not be infiltrated by the resin leaving holes in the section devoid of histological qualities.
For resin infiltration, first place the nerve segments in a one-to-one mix of embedding mixture and 100%acetone for 30 minutes. After 30 minutes, transfer the segments to a two-to-one mix of embedding mixture and 100%acetone for 30 minutes. Place the nerve segements in silicone rubber embedding molds and gently add the resin on top of nerves, making sure to cover the whole nerve segment while avoiding air bubbles.
Leave the resin to polymerize at 60 degrees Celsius overnight. Place a resin block with embedded nerve into the ultramicrotome holder with the trapezoidal side facing up. While viewing through the ultramicrotome scope, use a single-edged blade to trim excess resin surrounding the nerve tissue.
Do not penetrate the longitudinal surface of the nerve segment which is recognizable by its dark staining by osmium tetroxide. With a plain glass knife on the ultramicrotome make multiple cross sections to expose a uniform cross-section surface of the nerve. Once this is done, switch to a glass knife with the boat filled with distilled water at room temperature and adjust the ultramicrotome to one to two microns to cut thin sections.
Use a metal loop to transfer floating thin sections from the glass knife boat to a drop of deionized water on a glass slide. Another critical step is during the transfer of thin section from the glass knife to slide. These thin sections are very fragile and can break or fold.
After sectioning, dry the sections on the slides by passing a slide over a flame several times, making sure not to overheat the sections. Slides can be stored at room temperature for several days if not immediately staining with toluidine blue. To stain the nerve sections, use a plastic pipette or micropipetter to add a drop of toluidine blue solution on the top of the nerve sections.
After 20 to 30 seconds, rinse off all toluidine blue solution excess by gently dipping the slides into a jar of deionized water, repeating three to four times until sections are clear. Dry the slides for at least 15 minutes at 60 degrees Celsius or overnight at room temperature. After drying, add regular mounting medium and cover the sections with a coverslip.
Finally, examine the mounted sections under a light microscope. A 100X oil immersion lens is recommended for detailed images to calculate g-ratios. Sciatic nerve sections embedded in resin medium and stained with toluidine blue showed clear images with optimal resolution at increasing magnification from 10X to 20X, 40X, and 60X.
This method preserves nerve structure and allows for high-resolution, high-magnification imaging which facilitates the measurement of several parameters such as the g-ratio, which is the ratio of axon diameter, indicated by the arrow labeled B, to the total fiber diameter, labeled A.A variety of factors can lead to less than optimal peripheral nerve sections, including the presence of cracks in the section due to improper handling of the nerve. Holes can also occur in the section due to insufficient dehydration. Another possible error in the procedure is folding of the sections, which can be remedied by using inoculating loops for section transfer onto the glass slide.
Following this procedure other methods like axon quantification can be performed to answer additional questions like what's the degree of regeneration in a regenerative nerve? After watching this video you should have a good understanding of how to obtain one to two micron peripheral nerve sections to assess nerve morphometry.
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