The overall goal of this procedure is to visualize skin structures such as hair follicles and nerve endings in their entirety without physically sectioning the sample. This is accomplished by first collecting skin from various body regions, including the back hind paw and tail. The second step is to flatten and clean the skin samples.
Next skin samples are subjected to suitable fixation. The final step is to perform immunos staining or histo chemistry on the skin samples. Ultimately, imaging with a dissecting or confocal microscope is used to visualize the geometry of various skin structures.
The main advantage of this technique over existing methods like epidermal hormone mount preparation, is that it can be applied to skin in any location on the body and does not require separation of epidermis from the dermis. This method can help answer key questions in the study of plantar cell polarity. Since the two dimensional geometry of the skin and the oddly arrangement of hair follicles make the skin ideal system to study this process, Not only can this method provide insight into plant cell polarity, it can also be applied to study other aspects of skin development, such as the definition of somatosensory neuron types based on direct visualization of individual acts on upper morphologies.
Visual demonstration of this method is critical as skin dissection from some regions such as the feet and tail is difficult to learn, flattening them perfectly and avoiding wrinkles and overstretching are important. In this demonstration, dorsal skin will be collected from P three mice euthanized by isof fluorine. Inhalation use blades to make a horizontal cut from the base of the tail along each flank, passing on the dorsal side of the base of each limb proceeding lateral to the ears and ending at the nose.
Gently peeled the skin from the underlying tissue proceeding from posterior to anterior by holding the skin between gloved fingers so that it is not pinched by forceps. When peeling the skin at the level of the ears first, cut off the ears with a pair of scissors and proceed especially slowly as the skin can tear at this location. From this point onward, orient the skin with the inside facing up.
Pin the skin to S Guard with about 20 insect pins evenly spaced around the periphery. Do not overstretch the skin. Cover the skin with 10 to 20 milliliters of PBS using angled or curved forceps.
Dissect away the skin associated fat and connective tissue. Face the pointed end of the forceps horizontally to minimize the risk that the forceps will penetrate the skin. Extrude any air bubbles that have been trapped beneath the skin by gently rolling a cotton tipped applicator over the skin surface.
The next step is to fix the pin skin. If the skin will be used for melanin imaging or alkaline phosphatase histochemistry, add 20 milliliters per 10 centimeter cigar plate of freshly prepared 4%P-F-A-P-B-S or 10%buffered formin. If the skin will be used for immuno staining or visualizing a transgenic keratin, KRT 17 GFP add 20 milliliters per 10 centimeter sogar plate of freshly prepared 2%P-F-A-P-B-S gently rotate overnight in a cold room on the following day, wash in PBS for greater than 10 minutes.
The procedure for alkaline phosphatase histochemistry will not be demonstrated in this video. For melanin imaging, dehydrate the skin through a graded ethanol series one day for each step with gentle horizontal rotation at room temperature. Three days later, remove residual connective tissues from the dehydrated skin skin with the skin in 100%ethanol.
Remove the insect pins and transfer the skin to a 10 centimeter diameter glass dish. For the success of this procedure, the skin sample must be collected as a intact piece and flattened perfectly. Place two glass microscope slides on the skin to flatten it and prevent it from curling.
Add 20 milliliters of benzoyl benzoate and benzoyl alcohol or BBBA, which will rapidly harden the tissue over the next several hours. Lift the slides off the skin for a few seconds to allow the BBBA to wash over the skin. Foot skin for the analysis of hair follicle patterning is typically dissected from P one to P eight to animals After euthanasia, cut off the feet above the ankle joint and make a single straight cut along the ventral surface through the soles of the feet.
Gently peel the skin from the underlying tissue proceeding in a proximal to distal direction. Cut the digits to release the skin and pin the skin to sard with the inside surface facing up. There is minimal connective tissue on the foot.
Skin transgenic. KRT 17 GFP skin is now ready for imaging for melanin imaging. Fix and process the skin as demonstrated earlier for dorsal skin After euthanasia of the P 21 animal, remove the hair by rubbing hair remover.
Gel over the skin surface with a gloved finger and thumb and wait for 10 minutes. Wash the skin surface with tap water. Subsequently dissect and pin the foot skin to S guard as shown previously.
Fix with 1%P-F-A-P-B-S for 30 minutes at room temperature and then wash in PBS. To prepare tails skin, make a circular cut around the base of the tail and then a longitudinal slit that runs the length of the tail along its ventral face. Peel off the tail skin starting at the tip by firmly grasping the tip of the tailbone and or connective tissue with one pair of forceps and the skin with a second pair of forceps.
Pin the tail skin to SIL guard for the analysis of melanin distribution and hair follicle orientation. Fix and process the skin as demonstrated for dorsal skin for analysis of sebaceous glands prior to fixation. Cut the tails skin into a series of segments about 0.5 to one centimeter in length, and incubate the skin pieces in PBS five millimolar EDTA overnight at 37 degrees Celsius.
To weaken the dermis epidermis adhesion on the following day, gently peel the epidermis from the dermis. Pin the epidermis interface up to S guard and fix in 4%P-F-A-P-B-S for one hour at room temperature. Subsequently wash the epidermis with PBS and stain with oil red O as described in the protocol text.
A dissecting microscope is used to image dorsal foot or tails skins that are submerged in BBBA flattened beneath the glass slide and still in the glass dish. This approach minimizes BBBA contamination of the microscope. Illuminate the dish from below to minimize spatial variation in light intensity across the field of view.
Elevate the glass dish to a height of about 10 centimeters above the standard working surface of the dissecting microscope and place a diffusing plastic or glass plate over the light source. When imaging is done, return the skin to 100%ethanol for long-term storage at negative 20 degrees Celsius. To prepare alkaline phosphatase stained skin for imaging, place it between two glass plates of the type used for small protein gels.
Being careful to avoid introducing air bubbles between the plates and the skin. Carefully wick away any excess BBBA with a paper towel. Place this sandwich of plate skin plate on a microscope stage.
Use brightfield or DIC illumination with a 10 x objective and two micrometer or five micrometer Z steps. Use a mechanized XY stage to capture a montage of XY images that are stitched together to create a single three-dimensional gray scale dataset. Perform semi-automated tracing of axon arbors with any of a variety of software packages.
Brightfield imaging of skin flat mounts can be used to image a single cutaneous sensory arbor shown here with its traced image. Brightfield imaging also enables visualization of hair follicle patterns based on melanin pigmentation. These images show P one skin from wild type and frizzled six knockout mice and P seven skin from Frizzled six knockout mice.
Light microscopy imaging of skin flat mounts can be used to define the geometry of sebaceous glands and tails. Skin visualized with oil red O sebaceous glands are disorganized in a frizzled six knockout mouse compared to a wild type dorsal foot. Skin harvested from wild type and frizzled six knockout mice carrying A GFP tagged keratin KRT 17 GFP and stained with oil.
Red O revealed that the knockout skin has a hair whirl in its center. Confocal imaging of skin flat mounts can also define the geometry of hair follicles visualized with KRT 17 GFP and anti GFP immuno staining and erector pill. Eye muscles visualized with anti smoo muscle actin immuno staining.
Merkel cell clusters are visualized with anti cytokeratin eight or with am 1 43 dye uptake and its central hair follicle visualized with KRT 17 GFP Instead of being open toward the anterior, the Frizzled six knockout Merkel cell cluster forms a closed circle Once mastered a dissection can be done in approximately 30 minutes if it is performed properly and different standings can be done within a few days to a week. After watching this video, you should have a good understanding of how to collect skin from various regions of the body and how to stand them to visualize different skin structures in the hormone format. Full thickness, high resolution images of skin structures can be collected by standard high light microscopy and the confocal microscopy.
Don't forget that working with paraform aldehyde and BBBA can be extremely hazardous. Inhalation of vapors should be minimized by keeping these solutions in covered containers and skin contacts should be avoided. Do not allow BBBA to contact plastic.
