This article describes the procedure for cranial ring implantation on the mouse skull, which is essential for high-resolution imaging of the underlying bone marrow. The method ensures optimal visualization of specialized cells in their native environment.
For cranial ring implantation on the mouse skull, first, prep an anesthetized mouse in the prone position. Next, make a midline vertical incision to expose the calvarium – the top part of the skull that protects the cranial cavity enclosing the brain.
Now, carefully remove the underlying periosteum — a dense layer of vascular connective tissue – and any tissue debris for optimal imaging. Clean the exposed skull surface with saline.
Next, apply cyanoacrylate glue to the metal skull ring. Mount it over the desired area of the dry exposed skull.
Hold the ring in place to allow it to attach firmly to the skull. Keep the skull surface hydrated with saline. Secure the ring to the skull area with dental cement to prevent any leakage during subsequent imaging.
Fill the ring with saline, which acts as the immersion medium for high-resolution microscopy.
Transfer the skull ring-bearing mouse to a support plate affixed to the block holder. The mouse’s head is raised to prevent ring detachment from the skull.
Fix the ring onto the block holder to immobilize the mouse’s head and prevent respiratory-induced motion artifacts during imaging. Position the support and the mouse assembly for intravital imaging through the skull bone, which facilitates the visualization of specialized cells and their native environment in real-time in the underlying bone marrow.
Disinfect the scalp with 70% ethanol with a paper towel while removing the loose hair. Use sterile fine scissors and tweezers to make a T-shaped incision at the midline up to 1 centimeter in-between the ears on the scalp to expose the calvarium.
Expose the skull with tweezers. Then, use scissors and tweezers to carefully remove the periosteum. Use a sterile cotton swab soaked in physiological saline to remove all the periosteum and any debris or hair which could alter the imaging. Rinse the skull with saline to remove any blood traces, and rapidly dry the bone with a cotton swab.
Apply the glue gel to the ring. Place the ring on the exposed bone, and maintain it for a few seconds to allow the ring to firmly attach to the skull. Lightly moisten the skull with a saline-wetted cotton swab.
Prepare the silicon dental paste by carefully mixing the blue and yellow components at a 1 to 1 ratio. Seal the ring by applying the dental paste to prevent leakage during imaging. Carefully remove any dental paste or glue that may have entered the ring. Then fill the ring with saline to check the leakage.
Place the mouse on the support, and put a folded compress under the animal's head to raise the head and prevent detachment of the ring from the skull when attached to the holder. Screw the ring on the block holder. Place the support and mouse assembly in the heated microscope chamber.
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