Dissection of single skeletal muscle fibers for immunofluorescent and morphometric analyses of whole-mount neuromuscular junctions. This video will demonstrate a protocol to effectively and securely dissect EDL and soleus muscles with a repeatable approach. This video will also teach how to obtain whole-mount neuromuscular junctions with the aim of recognizing synaptic components with 3D integrity to allow an accurate morphometric analysis.
To dissect the muscles, these surgical instruments are needed. To start the procedure, place the animal with the abdomen facing upwards. Make an initial incision using a surgical blade between the toes towards the hind limbs.
Peel off the skin, pulling upward until the right knee is exposed. To find the EDL, follow the foot tendons to the annular ligament. This ligament circles two tendons.
Cut the ligament with the Uniband scissors between the two tendons. Identify the EDL tendon by lifting both and seeing which one makes the toes move upwards. Cut the tendon with scissors.
While holding the tendon with biological tweezers, begin to slowly separate the EDL muscle from the other hind limb muscles. Separation of the muscle without causing damage can be done by making various cuts on the rest of the lateral muscles to open a path between them, while holding and lifting the muscle from the splitting section of the EDL tendon. To completely isolate the muscle, cut the tendon that is attached to the rat knee using Uniband scissors.
Immerse the dissected muscle in the fixative solution, and leave it for 24 hours at four degrees Celsius. In general, it is useful to have an elongated muscle. To do this, use cardboard and staples to attach the muscle for fixation.
To isolate the soleus muscle, flip the animal. Through the skin, cut the calcaneal tendon using a surgical blade. With the help of the biological tweezers and Uniband scissors, separate the gastrocnemius muscle from the bones, creating a muscular lid.
The soleus will be on the internal side of the muscular lid. It can be identified because it is red and flat. With a pair of biological tweezers, reach and lift the soleus tendon that lies above the above the gastrocnemius.
Cut the tendon with Uniband scissors. Lift the whole muscle while cutting some of the weak attachment points. Finally, to completely free the soleus muscle, cut the soleus vesicle that forms the calcaneal tendon.
Repeat the fixation step as done with the EDL muscle. To use this method, it is important to secure the staples to the tendons and not to the muscle. Once the 24-hour period of fixation has passed, rinse the muscles in DPBS solution before beginning to isolate the muscle fibers.
In order to isolate the fibers, gently hold one of the tendons with one pair of biological tweezers. Then with the other pair of biological tweezers, begin to slowly pinch the tendon to separate the muscle fibers. To isolate the fibers, slowly pull upwards towards the opposite muscle tendon.
It will be necessary to repeat this action several times until obtaining multiple, small, isolated bundles. Once separated in small isolated bundles, place them carefully in a pre-treated silanized slide. It is necessary to keep all the fibers orderly so that they do not overlap.
At this point, the fibers can be left to air dry for 24 hours. After 24 hours, begin the immunostaining. To create a waterproof barrier, use a PAP pen to encircle the isolated muscle fibers in the pretreated slide.
This is an example of a high-resolution confocal image that can be obtained following the protocol described here. On the upper left, the post-synaptic element is shown, and on the upper right, the pre-synaptic element, while the bottom images show the synaptic components schematized in order to illustrate the parameters that will be used for morphometric analysis. The second slide shows the merging of the pre-and post-synaptic signals with the nuclei stain of the API.
Differences among the neuromuscular junctions of transgenic animals and non-transgenic animals are clearly visible. The morphometric analysis of neuromuscular junctions shows evidence of the post-synaptic signal in transgenic animals and appears to be more compact, mainly due to reduced neuromuscular junction total area. As shown here, the neuromuscular junction pre-synaptic area of the transgenic animal is reduced.
The smallest detected was the one found with anti-phosphorylated neurofilament signal, as shown in image B, for example. The last image shows the results pertaining to the status of whole-mount neuromuscular junctions. Using the coverage index, it was established that the transgenic neuromuscular junctions were found be partially denervated.
Also, it could determine that the coverage and nexus of the phosphorylated neurofilament is lower than obtained when a non-phosphorylated neurofilament is detected. The proposed protocol allows obtaining high-quality neuromuscular junction preparations from slow and fast muscle fibers. Pre-and post-synaptic neuromuscular junction components were identified by specific probes or antibodies, and then imaged in confocal or super-resolution confocal microscopy, enabling morphometric analysis at a micrometric scale.
