The overall goal of this histological and imaging protocol is to present a robust system that preserves the epitol antigenicity and structural integrity of skin, which allows for full exploitation of confocal imaging modalities. This method can help answer key questions where there is a requirement to examine the nature of any type of cell within a complex three dimensional tissue architecture. The main advantage of this technique compared to standard histological techniques is that it is robust and easy to perform.

Additionally, the protocol perfectly complements three dimensional analysis in confocal microscopy. Begin by trimming skin harvested from the dorsal medial region of a mouse into rectangular pieces. Each piece should be sized to fit in the bottom of a cryomold.

A one square centimeter area of dorsal skin which fits into a 22 millimeter by 30 millimeter by 20 millimeter cryomold is demonstrated here. After fixing, push the washed skin to the bottom of the OCT filled mold so that it lies flush with the bottom. Mark the hair follicle orientation on cryomold, as this determines the orientation of the cryo stat cut.

Transfer the cryo blocks onto a metal plate in a minus 80 degree freezer to prevent floating and dislocation of the tissue. Monitor the freezing process to maintain the orientation of the skin at the bottom of the cryomold, since unseen air bubbles can cause the skin to rise to the surface of the cryomold. Begin by fixing a frozen cryoblock to the stage of a cryostat with the hair follicle orientation, as indicated on the cryomold along the sectioning plane.

Proper adjustment of the exact hair follicle orientation on the cryostat is critical, since this step determines the section plane which generates skin slices with the entire length of hair follicles intact. Use the cryostat to cut a 100 micron section. Grasp the OCT around the embedded piece of skin with forceps and transfer the section out of the cryostat into the 100 millimeter culture filled with PBS.

At room temperature, the PBS will dissolve away the OCT, leaving skin slices that are easy to handle with forceps. After sectioning, use forceps to transfer the floating skin sections to the well of a 12 well plate containing 2.5 milliliters of PBS. Begin immunofluorescent labeling by first adding 500 microliters of PB buffer, to labeled 1.5 milliliter microcentrifuge tubes.

Carefully use forceps to transfer the skin slices from the 12 well plate into the tubes for blocking. Make sure that all skin slices are fully submerged. Place the microcentrifuge tubes on a see saw rocker at 10 oscillations per minute or less, for one hour at room temperature.

Label separate 1.5 milliliter microcentrifuge tubes for each skin slice, and add 500 microliters of PB buffer containing the appropriate amount of primary antibody. After one hour of blocking, transfer the skin slices into the freshly prepared tubes containing the antibodies. Incubate the slices at four degrees celsius overnight at a low speed on a rocker.

The next day, transfer the slices to 1.5 milliliter microcentrifuge tubes containing 500 microliters of PBS and wash for one hour at room temperature on a rocker. Then, transfer the slices into separate 1.5 milliliter microcentrifuge tubes containing 500 microliters of PB buffer with the appropriate concentration of secondary antibodies and DAPI. Incubate at room temperature for one hour with rocking, if desired, samples can be stored for up to four days at four degrees celsius until further processing.

Prior to imaging, transfer the skin slices to separate microcentrifuge tubes containing 500 microliters PBS to wash the secondary antibodies and DAPI. Then place a 22 milliliter by 50 milliliter cover slip onto a dark background under a dissecting microscope. Next, use a 1, 000 microliter pipette with the end of the tip cut off, to add one droplet of 100%glycerol onto the cover slip.

Transfer the skin slice from the microcentrifuge tube onto the glycerol droplet. Then, while looking through the dissecting microscope, use pointed forceps to carefully unwind the skin slices that are curled up by coaxing them back to their natural shape while floating in glycerol. Do not force the unnatural straightening of the slice, since this could cause damage to the tissue.

Once the entire length of the section is properly oriented and flattened on the cover slip. Mount the tissue on a regular microscope slide, this step will further straighten the skin slice. Image the in glycerol mounted skin sections within the next two days.

This image shows a 10 micron thick classically obtained skin cryo section labeled with integrin alpha six and integrin alpha eight, to visualize the epidermal compartment and directorial pili muscles respectively. This 100 micron thick 3D tissue cross section was labeled in the same way. The image shown is the maximum projections of a large z stack.

Detail from both images are compared side by side here, in the classical frozen sections, most hair follicles visualized with integrin alpha six were not sectioned along the entire length, generating predominantly incomplete hair follicles per section, as compared to horizontal whole mounts. Intact hair follicles and intact arrector pili muscles were quantified in both the classical and the horizontal home mount sections. One section for biological replicate was quantified, as seen here, the whole mount section has a higher proportion of intact follicles and arrector pili muscles.

Once mastered, this cryosectioning technique can be performed at speeds of more than 15 sections per minute, while achieving perfectly oriented samples if mounting was performed correctly. After watching this video, you should have a good understanding of how to process and analyze thick tissue cross sections in a three dimensional environment.