Whole-mount Confocal Microscopy for Adult Ear Skin: A Model System to Study Neuro-vascular Branching Morphogenesis and Immune Cell Distribution

The overall goal of this procedure is to visualize branching morphogenesis and patterning of peripheral nerves and blood vessels, as well as immune cell distribution. This method can help answer key questions in the fields of neurobiology, vascular biology, and immunology by illuminating morphological abnormalities in peripheral nerves and blood vessels, as well as inflammation. The main strength of this technique is that we can visualize the cellular components of peripheral nerves, blood vessels, and immune cells in the skin over its entire depth and can compare three-dimensional architecture on nervous and vascular systems.

This method can provide insight into neuro-vasculature structures and immune cell distribution in juvenile to adult mice under normal and pathology conditions. After euthanizing adult mice according to the text protocol, dissect the ear from the base, and place it in a 35-by-10-millimeter Petri dish containing two milliliters of HBSS. Then, use scissors to briefly trim the hairs.

Carefully peel the posterior and anterior skin away from the intervening cartilage. Transfer each section of skin separately to a 24-well plate containing one milliliter of ice-cold, fresh 4%PFA in PBS per well. Then, flatten the skin in the PFA.

Next, fix the posterior and anterior skin with cartilage at four degrees Celsius for one hour. Then, use one milliliter of PBS to wash the skin three times for five minutes with gentle mixing at room temperature. Transfer the skin with cartilage to the bottom of a 35-by-10-millimeter Petri dish.

Use fine curved forceps to peel off the cartilage from the anterior skin. Then, cut off the base region, which is folded and has adipose and connective tissues. Using the same forceps, carefully remove the hairs, adipose tissue, and connective tissue from the inside of the posterior skin.

Use PBS to keep the skin wet. To carry out whole-mount immunohistochemical staining, transfer the posterior and anterior skin to a 24-well plate containing one milliliter of 10%heat-inactivated serum blocking buffer per well. Incubate the skin with gentle mixing at room temperature for 30 minutes.

Dilute the primary antibodies in the blocking buffer as described in the text protocol, and add 150 microliters of primary antibodies in blocking buffer to the well. Then, transfer the posterior and anterior skin to new wells containing primary antibodies. Incubate the skin with gentle mixing at four degrees Celsius overnight.

The following day, after transferring the skin to new wells or aspirating the primary antibodies, add one milliliter of 2%serum in PBS with 0.2%Triton X-100. Wash the samples with gentle mixing at room temperature for 15 minutes. Repeat the wash two more times.

In the meantime, prepare secondary antibodies in 10%blocking buffer and filter the antibody solutions through a 0.22-micrometer PVDF membrane syringe filter connected to a one-milliliter syringe. Centrifuge the solution at 13, 000 times gravity for 10 minutes to remove aggregated particles of the secondary antibodies from the blocking buffer, adding 150 microliters of secondary antibodies in blocking buffer to the new well. Transfer the skin to the well containing secondary antibodies.

Wrap the 24-well plate in aluminum foil to protect it from light, and incubate the skin with gentle mixing at room temperature for one hour. Add one milliliter of 2%serum washing buffer. Then, transfer the posterior and anterior skin to the wells containing washing buffer.

Wrap the plate, and wash the samples with gentle mixing at room temperature for 15 minutes. Repeat the wash two more times. Place the skin on the bottom of a 35-by-10-millimeter Petri dish.

Then, under a stereo microscope with low illumination to minimize photo bleaching, use fine curved forceps to carefully remove the hairs, adipose tissue, connective tissue, dust, and fibers from the inside of the skin. Use PBS to keep the skin wet. With forceps, transfer the posterior and anterior skin, with the inside facing up, on an adhesive microscope slide.

Use forceps to flatten the skin. Mount the skin in a liquid anti-fade mounting medium to avoid photo-bleaching and preserve fluorescent signals. Make sure that no air bubbles are on or around the skin.

Store the stained skin sample slides in the dark overnight at room temperature to allow the mounting medium to get firm. Then, use nail polish to seal the coverslip to the slide, and store it at four degrees Celsius for long-term storage. To carry out confocal microscopy, set up appropriate lasers for fluorophores.

A confocal microscope with three laser sources is used in this experiment. Image the samples under a 10X objective, and use the sequential scan tool, which simultaneously excites triple-stained samples, to avoid and reduce any overlap. In this experiment, adult mouse posterior ear skin and anterior ear skin were immunostained with antibodies to alpha-SMA, Tuj1, and PECAM-1.

The posterior skin was immunostained to study neuroimmune distribution using antibodies to CD11b and MBP, together with Tuj1. Finally, as shown here, distribution of CD11b-positive inflammatory cells, including macrophages, was detected at a single-cell resolution. While attempting this procedure, it's important to remember to handle your skin with care and to gently and slowly remove the hairs, adipose tissue, and connective tissue from the inside of the ear skin before mounting.

This technique will pave the way for researchers to study branching morphogenesis and patterning of peripheral nerves and blood vessels, as well as three-dimensional distribution of skin components, including immune cells in juvenile to adult mouse models. Thanks for watching. Good luck with your experiments.