Demonstrating Hairy and Glabrous Skin Innervation in a 3D Pattern Using Multiple Fluorescent Staining and Tissue Clearing Approaches

Our protocol provided a feasible method to observe the sync section using confocal microscope, which could ensure precision in the observation of the nerve fibers. Simple and easy tissue cleaning protocol is a main advantage of this technique. The tissue cleaning process is efficient and it takes about 30 minutes to one hour.

Conduct perfusion on the euthanized rat as mentioned in the text manuscript. Use a scalpel to remove the skin of the dorsum and sole from the hind paw. For samples requiring a frozen section, post fix the tissues in 4%from aldehyde in 0.1 molar PB for two hours.

After washing, embed the skin tissues into 2%agarose at 37 degrees Celsius and place them inside the ice box for cooling. Fix the mounted tissue on the vibratory microtone with ice water and slice them at a thickness of 500 micrometers in the transversal direction. After slicing, remove the agarose from the sections and store them in a six well plate with 1x PBS.

Incubate the tissue sections in a solution of 2%triton X-100 in 1x PBS overnight at four degrees Celsius. Place the tissue sections into the blocking buffer and rotate at 72 rotations per minute on the shaker overnight at four degrees Celsius. Transfer the tissue sections into the solution containing the primary antibodies of mouse monoclonal anti-CGRP and sheep polyclonal anti-LYVE-1 antibody in dilution buffer in the micro centrifuge tube.

Then rotate on the shaker for two days at four degrees Celsius. Wash the tissue sections twice with washing buffer at room temperature, then keep them on the shaker overnight at four degrees Celsius in the washing buffer. The next day, transfer the tissue sections into the mixed solution containing the secondary antibodies in a micro centrifuge tube and rotate at 72 rotations per minute on the shaker for five hours at four degrees Celsius.

Wash the tissue sections in a six well plate with washing buffer twice for one hour at room temperature. Keep the tissue sections in washing buffer on the shaker overnight at four degrees Celsius. Transfer the tissue sections into the tissue clearing reagent and rotate at 60 rotations per minute on the shaker gently for one hour at room temperature.

Mount the clear tissues on the slide then circle the tissues with a spacer and stick the gap with fresh tissue clearing reagent and cover slip. The 3D pattern demonstrated that the CGRP positive nerve fibers passed through the subcutaneous tissue and dermis to the epidermis. These nerve fibers ran in bundles in the subcutaneous tissue branched within the dermis and terminated in the epidermis.

In contrast, phalloidin in positive blood vessels and LYVE-1 positive lymphatic vessels are distributed in the subcutaneous tissue and dermis. Generally the CGRP positive nerve fibers ran parallel to or surrounded the blood vessels and lymphatic vessels forming a 3D network in the hairy and glabrous skin. With the conventional approach, although the nerve fibers blood vessels and lymphatic vessels were clearly labeled with CGRP phalloidin and LYVE-1 in the thin sections, the observation of these structures was limited by the slice thickness and cannot be observed completely.

In the section with a thickness of 30 micrometers, there was no difference between hairy skin and glabrous skin in the surface area of CGRP positive nerve fibers. In the 300 micrometers thick tissue sections, compared with hairy skin, the surface area of CGRP positive nerve fibers of glabrous skin was significantly increased. It's important to use the cleaning buffer with high osmotic pressure after antibody incubation.

Several times, the cleaning time required for the immuno fluorescence staining is necessary. Following this procedure, we believe there is a possibility of staining and observation of other tissues and organs in six sections also exist. This technique could assist neuroscience researchers encountering the surface area and the state of neuro fibers.

It also helps us to observe the innovation of tissues and organs.