Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

The transverse ADOS muscle is a thin flat muscle making up the deepest layer of the abdominal musculature. It is attached to the midline via the linear alpha and to the deep surface of the thoracic cage. In this video, we will dissect the superior part of this muscle and use immunofluorescence to visualize innovation patterns and neuromuscular junction structure.

For this dissection, you will need These tools, which are listed separately in the attached protocol. After euthanizing the mice by an appropriate method spray with 70%ethanol to minimize the spread of fur. Place the mouse in its back and make an incision through the skin at the level of the hip.

Being careful not to cut through the abdominal musculature. Continue this incision all the way around the mouse. After completing the incision, pull the skin up over the head of the mouse.

Again, being careful not to rupture the abdominal musculature. Cut through the Abdominal muscles and continue the incision until you reach the vertebral column. At this point, cut upwards through the ribs until you're above the sternum, and take care to release the diaphragm from the thoracic cage.

Repeat this process on the other side of the mouse. At this point, cut through the ribs and sternum and transfer the ribs with attached muscles to a algar coated dissection dish. Pin out the muscle using insect dissection pins.

Fix your tissue in 4%PFA and leave on a rocking platform For 15 minutes. Here is an example Of what your pinned out muscle should look like. On the left hand side of the image, the superficial muscles can be seen, which include the external oblique outlined in blue and the rectus abdominis outlined in red.

This layer has been peeled back on the right hand side of the image to reveal the internal oblique outlined in yellow and it transverses abdominis outlined in green. It is our aim to dissect the superior part of the transverses abdominis muscle. Shown here in the solid green triangle under a dissection microscope, cut through the external oblique muscle at a point where it covers The ribs, then cut down through the external oblique, but above the transverses abs Cut down until you see the start of the internal oblique muscle seen here as tangential muscle fibers on the left hand side of The image.

Remove the blood Vessel by gently peeling it from the surface of the muscle. Then use blunt dissection to separate the transverse abdominis from the overlying rib. Cut around the margins of the muscle, being sure to include the part underneath the rib.

Remove the rib and transfer the muscle to a 24 well plate for immunofluorescence. Perform all Immunofluorescence procedures in a 24 well plate. Using a fine tip pipet and follow the protocol within this manuscript.

Make sure you use the reagents detailed in this protocol. After immunofluorescent staining mount muscles on glass slides and fluorescent Mounting media, use Forceps to gently unfold the muscle, making sure it is flat before cover slipping. Make sure you press down Firmly to flatten out the muscle during mounting As shown in this image, this protocol can be used to perform whole mount staining of the transverses abdominis muscle to visualize neuromuscular junctions and innovation patterns.

In this image, axons and synaptic terminals are shown in green, marked with antibodies against SB two and neurofilament and motor pls as shown in red, labeled as alpha bunger toxin. Confocal microscopy Z Series can now be used to get high resolution images of neuromuscular junction morphology. This protocol can be applied to study neuromuscular junction pathology in mouse models.

For example, these images, so neuromuscular junction pathology in a mouse model of spinal muscular atrophy. Note, there is evidence of full or partial denervation indicated by white or purple arrows respectively, terminal sprouting shown by blue arrows and presynaptic swelling marked by yellow arrows. Such analysis can give important insight into the pathologic process in motor neuron diseases.