Axolotl Tissue Mounting and Processing for Atomic Force Microscopy Measurements in Regenerating and Intact Limb Tissues

To prepare one-centimeter long cylinders of one centimeter diameter, heat a cardboard cutter under a Bunsen burner flame, and cut a 15-milliliter tube with the heated blade. Cover one side of the cylinders with Parafilm pieces. After anesthethetizing the axolotl and making sure it is unresponsive to tactile stimuli, orient the limb perpendicular to the body axis.

Amputate the limb immediately distal to the calcified area of the zeugopodial region. Return the axolotl to a tank containing fresh holding water with analgesics. After 24 hours, transfer the axolotl back to fresh holding water and allow the limb to regenerate until the desired stage of interest.

After placing the anesthetized axolotl under a stereo microscope, measure the length of the limb at the desired stage of regeneration for histolysis and condensing cartilage stages. After collecting the limbs, place them in a Petri dish for dissection. Under a stereo microscope, dissect the limbs with a scalpel at the elbow level, and place them in a Petri dish containing APBS solution.

Arrange the Pasteur pipettes and the thermoblock set at 37 degrees Celsius with agarose aliquots on the working platform. Remove the cold block from the minus 20 degrees Celsius freezer and place the cylinder with the Parafilm-covered end facing down on top of it. Place the limb on a clean plate and gently remove excess liquid from the dissected limb with tissue paper.

Add the melted low melting point agarose on top, and briefly move the limb around in the agarose to displace any remaining APBS from the skin surface. Quickly place the limb inside the cylinder, ensuring it is oriented vertically with the area of interest facing upwards. Add low melting point agarose inside the cylinder until the tissue is fully covered.

Next, remove the cylinder from the cold block and allow the agarose to solidify for 30 seconds. After solidification, remove the Parafilm from the bottom of the cylinder and attach the agarose-containing tissue to the vibratome stage with cyanoacrylate glue. Then, remove any excess surrounding agarose with a scalpel.

Submerge the stage in APBS solution for sectioning. Begin sectioning the agarose in 100-micrometer steps until the tissue tip is reached. Then, section until the distal portion of tissue is removed.

Using a razor blade, carefully remove the tissue-containing agarose block from the vibratome stage. Immediately, attach the block to a 35-millimeter plastic Petri dish with surgical tissue adhesive glue. Add approximately two milliliters of culture medium to cover the tissue.