This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.
Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called "horizontal whole mount," is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 µm-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.
The revolution of microscopic imaging equipment provides for sophisticated, high-resolution imaging instruments. However, when acquiring a microscopic image of a complete three-dimensional (3D) tissue cross-section, specimen preparation presents considerable challenges and can be the limiting factor in defining image quality. Each separate step deserves careful consideration in order to preserve tissue morphology and the antigenicity of target proteins, to minimize processing-induced artefacts, and to maximize the final image quality. For example, the traditional analysis of skin requires an image with a view of the epidermis and dermis, with hair follicles that are properly oriented, allowing for the anatomical analysis of stem cell compartment contributions to skin homeostasis1,2. This requires thorough concentration on how the skin is embedded and sectioned. Importantly, hair follicles can be thicker than 100 µm, which greatly surpasses the standard paraffin or frozen section thickness, resulting in a lower standard of analysis compared to whole mounts or thick cross-sections 3,4,5.
Taken together, each step of specimen preparation for microscopic analysis is a critical determinant that will affect image analysis. Here, a novel processing protocol for thick, 3D tissue cross-section analysis, which we call "horizontal whole mount," is presented. The protocol highly preserves antigenicity and enables the full exploitation of thick sections of skin by using standard confocal imaging equipment. This is a complete guide to using skin for thick tissue cross-section processing and imaging, including tissue harvest and paraformaldehyde (PFA)-assisted cryopreservation (step 1), the generation of 100 µm-thick tissue cross-sections with a cryostat (step 2), and immunofluorescent labeling and mounting (steps 3 and 4). The representative results compare confocal images of two distinct histological preparation techniques—classical cryosectioning and thick, 3D tissue cross-sectioning—highlighting the advantages of "horizontal whole mounts" for the potential user of this protocol.
All animal experiments were subject to local ethical approval and performed under the terms of a UK government Home Office license.
1. Skin Harvest and Cryopreservation
Figure 1. Harvest and fixation of mouse skin.
(a) Skin tissue was harvested from the dorsomedial region of the animal cadaver. Hair follicles in this region are evenly spaced and aligned and therefore allow for optimal orientation during sectioning, as indicated by the arrows. (b) After cutting squares of an appropriate size that fits into the cryomold, the skin tissue was fixed in 4% PFA for 15 min and washed two times in PBS for 5 min each. Please click here to view a larger version of this figure.
2. Thick Tissue Cross-sectioning
Figure 2. Embedding, cryopreservation, and sectioning.
(a) Marking the hair follicle (HF) direction on the cryomold, indicated by the black arrows, is important for proper orientation during cryosectioning. (b) The section plane needs to be aligned with the hair follicle orientation to generate sections in which the complete length of the hair follicles stays intact.(c) Sections were cut per the hair follicle orientation that was indicated by the black arrows on the cryomold. (d) The thick tissue cross-sections were collected with metal forceps and (e) transferred into a 100 mm culture dish containing 1x PBS. (f) At room temperature, the PBS dissolves away the O.C.T. compound that surrounds the thick tissue cross-sections, as indicated by the white arrows. The sections then float freely in the PBS. Please click here to view a larger version of this figure.
3. Immunofluorescent labeling.
Figure 3. Immunofluorescent labeling and mounting.
(a) The floating tissue cross-sections can be stored in 12-well plates for at least two days at 4 °C. Before immunofluorescent (IF) labeling, transfer the tissue cross-sections of interest into 1.5 mL microcentrifuge tubes containing PB buffer for blocking, as indicated by arrow 1. For IF labeling, adhere to the multi-step procedure elaborated upon in step 3. Each part of step 3 requires the careful transfer of the tissue cross-sections into freshly prepared microcentrifuge tubes containing primary antibody solution, secondary antibody solution, or washing buffer, which is indicated by arrow 2. (b) After IF labeling, the tissue cross-sections are unraveled and flattened in a droplet of glycerol, using the assistance of a dissecting microscope. (c) Once the tissue cross-section is entirely flattened onto the bottom of a cover slip, a regular microscope slide is used to mount the section. Please click here to view a larger version of this figure.
4. Mounting for Microscopic Visualization
To emphasize the advantages of our technique, we compared our thick, 3D tissue cross-sectioning technique, "horizontal whole mount," to classical frozen sections. Classical frozen sections were cut as previously described5. To provide a visual structure for the epidermis in te microscopic images, we immunostained for integrin alpha-6 (Itga6), which is a component that anchors epidermal cells to the underlying basement membrane6. We also labeled the arrector pili muscle (APM), which is responsible for piloerection (also known as "goosebumps"), with integrin alpha-87. In the classical frozen sections, most hair follicles visualized with Itga6 were not sectioned along the entire length, generating predominantly incomplete hair follicles per section, as compared to horizontal whole mounts (Figure 4a-4d). Thick tissue cross-sections make it possible to acquire more Z-stack layers compared to conventional 10-µm sections, allowing for a more complete 3D image. This becomes even more apparent when studying the integrity of APMs, which are associated with the hair follicles and the overlying basement membrane. In classical cryosections, the vast portion of APMs were fractioned (Figure 4a-4d). Additionally, the tissue integrity of the hypodermal compartment is preserved in horizontal whole mounts, as compared to the destruction of adipocytes when cryosections are attached to warm glass slides, which is a well-known freeze-thawing artifact (Figure 4a-b, compare hypodermal regions)8.
Figure 4. Horizontal whole mount compared to a classical cryosection.
(a) Classically obtained skin cryosections 10 µm thick and (b) 100 µm-thick 3D tissue cross-sections were labeled with integrin alpha-6 (Itga6) and integrin alpha-8 (Itga8) to visualize the epidermal compartment and arrector pili muscles, respectively. The images of the thick tissue cross-sections are represented as maximum projections of a large Z stack. The white frames indicate the areas that are enlarged, displayed in (c) the classical and (d) horizontal whole mount sections. (e) Intact hair follicles and (f) intact arrector pili muscles were quantified in both the classical and the horizontal whole-mount sections. Scale bars indicate 100 µm. The data is represented as Mean ± Standard Error of the Mean (SEM). One section per biological replicate (n=3) was quantified. Unpaired t-test *P<0.05, ***P<0.0005. Please click here to view a larger version of this figure.
Here, we present the “horizontal whole mount” technique, which has several advantages over classical frozen sections. One advantage of our technique is that it can easily be performed with standard histology equipment and a confocal microscope. Second, the tissue integrity is preserved in a superior manner when compared to standard cryosections. For example, adipocytes within the hypodermal layer (i.e., dermal white adipocytes (DWAT))9 are severely disrupted in standard cryosections, which makes studying tissues that are adipose-laden impossible. Our technique allows for the full analysis of the adipocytes in this layer and may be adapted to other tissues with high adipocyte contents. Furthermore, this allows for the improved detection of mesenchymal cells in lineage-tracing studies 2,10.
Standard cryosections, by their very nature, can never reveal the true 3D aspects of a tissue through confocal microscopy. This means that thicker sections are advantageous in the analysis of skin, since hair follicles, as well as their substructures, can traverse a depth that exceeds the standard thickness of 10 µm used in classical crysosections. We are intrigued that other tissues, such as the intestine and brain, for example, possess similar challenges with regard to viewing the tissue as a 3D structure2,7,11,12,13. Intriguingly, our technique has already been shown to be beneficial for use in certain applications in the intestines14. We believe that our horizontal whole-mount protocol has the potential be applied to any other tissue requiring 3D analysis.
The authors have nothing to disclose.
The authors acknowledge sponsorship from Thermo-Fisher Scientific and thank the Nikon Imaging Centre at Kings College London for support during confocal image acquisition.
PBS | homemade | ||
Gelatin, from cold water fish skin | Sigma | G7765 | 250ML |
Glycerol | BDH Laboratory Supplies | 444482V | |
O.C.T. compound | VWR chemicals | 361603E | |
Peel-A-Way embedding molds | Sigma | E6032-1CS | Square S-22 |
Non-Fat Powdered Milk | Bio Basic Inc. | NB0669 | |
Triton X-100 | Sigma | T9284 | 500ML |
4′,6-Diamidino-2-phenylindole, dilactate (DAPI) | Invitrogen | D3571 | |
FITC Rat Anti-Human CD49f | BD Pharmingen | 555735 | |
Mouse/Rat Integrin alpha 8 Antibody | R&D Systems | AF4076 | |
Alexa Fluor 488 donkey anti-rat IgG | Life Technologies | A21208 | |
Alexa Fluor 555 donkey anti-goat IgG | Life Technologies | A21432 | |
CryoStar NX70 | Thermo Fisher Scientific | ||
100mm Culture dishes | |||
Disposable scalpels | Swann-Morton | Ref 0501 | |
pointed metal forceps | |||
1.5 ml microcentrifuge tubes | VWR | 211-2130 | |
12 Well plates | Sigma | CLS3513 | |
Dissecting microscope | Nikon | ||
20 ul and 1000 ul Pipette | Gilson | ||
1000µl XL Graduated TipOne Filter Tip (Sterile) | Star Lab | S1122-1830 | |
20µl Bevelled TipOne Filter Tip (Sterile) | Star Lab | S1120-1810 | |
Rocking shaker plate | |||
Microscope slides, menzel Glaeser | Thermos Scientific | 631-9483 | Superfrost Plus |
Cover Glasses, Menzel Glaeser | Thermos Scientific | MENZBB024060AB | 24 x 60 mm |
Confocal microscope | Nikon |