The authors acknowledge sponsorship from Thermo-Fisher Scientific and thank the Nikon Imaging Centre at Kings College London for support during confocal image acquisition.

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
<ol>
	<li>Preparations.
	<ol>
		<li>Prepare one 100-mm culture dish with 25 mL of 4% PFA and two 100-mm culture dishes with 25 mL of 1x phosphate-buffered saline (PBS).</li>
		<li>Fill rectangular peel-away cryomolds by two-thirds with optimal cutting temperature compound (O.C.T.).</li>
		<li>Place a metal plate, on which the cryoblocks can be placed in a later step, into the -80 &#176;C freezer.</li>
	</ol>
	</li>
	<li>Skin harvesting, fixation, and cryopreservation.
	<ol>
		<li>Clip the dorsal region of the animal cadaver with a dry electric shaver. 
		NOTE: In this example, postnatal day 21 wildtype mice were used.</li>
		<li>Harvest the areas of interest on the skin. 
		NOTE: The dorsomedial region of mouse skin (Figure 1a) contains the highest percentage of hair follicles that are evenly spaced and aligned, which allows for optimal orientation for sectioning. The removal of underlying non-dermal tissue is not necessary but can be performed if required.</li>
		<li>Trim the harvested skin into rectangular pieces of appropriate size to fit into the bottom of the cryomold, taking the directional growth of the hair follicle grain into account. 
		NOTE: Smaller skin slices might be easier to handle for novices, since they are less likely to tangle during the incubation and mounting process. The example shown here is a ~1 cm2&#160;area of dorsal skin, which fits into a 22 x 30 x 20 mm cryomold (Figure 2a).</li>
		<li>Fix the skin at room temperature in 25 mL of 4% PFA for 10-30 min, depending upon the thickness of the skin samples (Figure 1b).</li>
		<li>Wash the skin samples two times in 25 mL of PBS for at least 5 min each (Figure 1b).</li>
		<li>Dab the skin samples on a paper towel to carefully drain the tissue of excess PBS, which can result in crystallization during the freezing process and can affect cryosectioning results. 
		NOTE: A standard sucrose gradient is not required. However, this may also be incorporated into the protocol at the user&#39;s discretion.</li>
		<li>Be aware of the orientation of the hair follicles for each skin sample. Use a dissecting microscope for visual assistance (especially anyone performing the protocol for the first time). Insert the skin sample into the O.C.T.-filled cryomold and equilibrate all areas of the skin with O.C.T. by removing any air bubbles attached to the surface of the clipped hair using forceps ( Figures 2a and 2b).</li>
		<li>Push the skin to the bottom of the O.C.T. filled block so that it lies flush with the bottom. 
		NOTE: The skin can be oriented in any direction, as long as the grain of the hair follicle is noted for proper cutting procedures. The cryomolds will be re-oriented when the blocks are attached to the cryostat for cryosectioning. Mark the hair follicle orientation on the cryomold, since this step determines the subsequent orientation of the cryostat cut.</li>
		<li>Transfer the cryoblocks onto the metal plate in the -80 &#176;C freezer to avoid floating and dislocation of the tissue.</li>
		<li>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. 
		NOTE: Cryomolds with frozen tissue can be stored for more than a year at -80 &#176;C and can be reused for additional sections.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56106/56106fig1.jpg" /> 
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. <a href="http://cloudfront.jove.com/files/ftp_upload/56106/56106fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Thick Tissue Cross-sectioning
<ol>
	<li>Preparation and tissue orientation to mount on the cryostat.
	<ol>
		<li>Prepare a 100-mm culture dish with 15 mL of PBS. Place it on an easily-accessible area on the cryostat. In addition, prepare a 12-well plate with 2.5 mL of PBS per well; label according to the samples for the long-term storage of the sections at 4 &#176;C. Use forceps to handle the sections.</li>
		<li>Adjust the temperature of the cryostat to -20 &#176;C. 
		NOTE: The temperature can affect sectioning, but a good guide is to start at -20 &#176;C.</li>
		<li>To obtain sections approximately two hair follicles thick, adjust the cryostat to cut sections 150 &#181;m thick. 
		NOTE: The thickness of the section can be varied, depending upon the needs of the user and the limitations of the microscope that will be used for analysis. 
		NOTE: The orientation of the sample is critical to obtaining skin sections with hair follicles in the appropriate orientation. This is achieved by mounting properly on the cryostat block. Make sure that the section plane is parallel to the hair follicle orientation (Figure 2c). As mentioned previously, the correct orientation of the section plane in the skin sample is a crucial step to determining the quality of the images that will be acquired in a later step.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56106/56106fig2.jpg" /> 
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. <a href="http://cloudfront.jove.com/files/ftp_upload/56106/56106fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Cryosectioning.
	<ol>
		<li>Cut a section using the cryostat. Use forceps to collect the O.C.T. that contains the embedded piece of skin (Figure 2d). 
		NOTE: Use a cryostat that enables independent movement and adjustment along the X, Y, and Z axes for optimal specimen positioning. This allows for the generation of ideally aligned tissue cross-sections.</li>
		<li>Transfer the section out of the cryostat into the 100 mm culture dish filled with PBS and continue with the next slice. Do not collect the samples on a slide (Figure 2e). 
		NOTE: At room temperature, the PBS will dissolve away the O.C.T., leaving skin slices that are easy to handle with forceps (Figure 2f). 
		NOTE: Fresh PBS may be needed in the 100 mm culture dish after dissolving many tissue sections 100 &#181;m thick and can be changed accordingly.</li>
		<li>Use forceps to transfer the floating skin sections to the correctly labeled well in a 12 well plate filled with 2.5 mL of PBS (Figure 3a, left). 
		NOTE: At 4 &#176;C, the samples can be stored for at least two days. For the long-term storage of skin-containing O.C.T. blocks after sectioning, seal the cutting surface with a droplet of fresh O.C.T. After freezing of the O.C.T. droplet, wrap the used O.C.T. block in parafilm and place it back in the -80 &#176;C freezer.</li>
	</ol>
	</li>
</ol>
3. Immunofluorescent labeling.
<ol>
	<li>Prepare the PB buffer (PBS supplemented with 0.5% skim milk powder, 0.25% fish skin gelatin, and 0.5% Triton X-100) at least 2 h in advance, as described previously5. 
	NOTE: Sodium azide can be added to PB buffer for antibody preservation for the repeated use of the staining buffer.</li>
	<li>Label 1.5 mL microcentrifuge tubes and add 500 &#181;L of PB buffer per tube. Carefully use forceps to transfer the skin slices from the PBS into separate tubes containing the PB buffer for blocking (Figure 3a, right). Make sure that all skin slices are fully submerged. Place the microcentrifuge tubes on a see-saw rocker at speeds no higher than 10 oscillations per minute, which should not disrupt the tissue integrity, for 1 h at room temperature. 
	NOTE: It is critical that the speed does not exceed 10 oscillations per minute on the see-saw rocker, since it will induce tangling.
	<ol>
		<li>While it is possible to add more than one slice per microcentrifuge tube, to save antibodies, only place one slice per tube. To decrease antibody usage, use a volume of 250 &#181;L. 
		NOTE: If needed, substitute microcentrifuge tubes with, for example, 96-well plates. However, the placement of the thick tissue cross-sections into 1.5-mL microcentrifuge tubes allows for the most effective antibody penetration into the tissue due to the enhanced liquid perturbation.</li>
	</ol>
	</li>
	<li>Label separate 1.5 mL microcentrifuge tubes for each skin slice and add 500 &#181;L of PB buffer and the appropriate amount of primary antibody. After 1 h of blocking, transfer the skin slices into the freshly prepared tubes containing the antibodies. Incubate the slices at 4 &#176;C overnight. 
	NOTE: In this example, the following primary antibodies were used: FITC rat anti-human CD49f at a concentration of 1:50 and goat anti-mouse/rat integrin alpha 8 at a concentration of 1:100.</li>
	<li>The next day, prepare two separate 1.5 mL microcentrifuge tubes containing 500 &#181;L of PBS per sample. Wash the skin slices two times for 1 h at room temperature.</li>
	<li>Prepare separate 1.5 mL microcentrifuge tubes containing 500 &#181;L of PB buffer with the appropriate concentration of applicable secondary antibodies and 4&#39;,6-diamidino-2-phenylindole (DAPI). 
	NOTE: The secondary antibodies used in this example are Alexa Fluor 488 donkey anti-rat IgG and Alexa Fluor 555 donkey anti-goat IgG at a concentration of 1:500. DAPI was used at a concentration of 1:100.</li>
	<li>Carefully transfer the skin samples into the PB buffer, which contains the secondary antibody and DAPI, and incubate the skin slices at room temperature for 1 h at a low speed on a rotator or shaker.</li>
	<li>Store the slices at 4 &#176;C in the PB buffer containing the secondary antibody and DAPI for up to four days, and possibly longer if sodium azide is added to the PBS.</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56106/56106fig3.jpg" /> 
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 &#176;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. <a href="http://cloudfront.jove.com/files/ftp_upload/56106/56106fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Mounting for Microscopic Visualization
<ol>
	<li>Prior to imaging, transfer the skin slices to separate microcentrifuge tubes containing 500 &#181;L of PBS to wash away the secondary antibodies and DAPI.</li>
	<li>Use a 1,000 &#181;L pipet, but cut off the first 0.5 cm of the pipette tip to enable the proper pipetting of the highly viscous glycerol. Place a 22 x 50 mm coverslip onto a dark background under a dissecting microscope (Figure 3b).</li>
	<li>Add one droplet of 100% glycerol onto the cover slip (Figure 3b). Transfer the skin slice from the microcentrifuge tube onto the glycerol droplet. Use the dissecting microscope and pointed forceps to carefully unwind the skin slices that are curled up. 
	NOTE: As the slice floats in the glycerol droplet, it can be untangled by coaxing the natural propensity of the tissue to return to its normal shape. Do not force the unnatural straightening of the slice, since this could cause damage to the tissue.</li>
	<li>Mount the tissue once the entire length of the skin section is properly oriented and flattened on the cover slip; use a regular microscope slide. This step will further straighten the skin slice. 
	NOTE: Avoid air entrapment (Figure 3c).</li>
	<li>Image the in glycerol-mounted skin sections within the next two days. 
	NOTE: Prolonged storage will negatively influence the tissue and imaging quality. In this example, all images were acquired with an upright confocal microscope using a 20x objective.</li>
</ol>

<ol><li>Plikus, M. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cyclic+dermal+BMP+signalling+regulates+stem+cell+activation+during+hair+regeneration%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Cyclic dermal BMP signalling regulates stem cell activation during hair regeneration.</a> Nature. 451 (7176), 340-344 (2008).</li>
<li>Zhang, Y. V., Cheong, J., Ciapurin, N., McDermitt, D. J., Tumbar, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distinct+self-renewal+and+differentiation+phases+in+the+niche+of+infrequently+dividing+hair+follicle+stem+cells%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Distinct self-renewal and differentiation phases in the niche of infrequently dividing hair follicle stem cells.</a> Cell Stem Cell. 5 (3), 267-278 (2009).</li>
<li>Jensen, U. B., Lowell, S., Watt, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+spatial+relationship+between+stem+cells+and+their+progeny+in+the+basal+layer+of+human+epidermis%3A+a+new+view+based+on+whole-mount+labelling+and+lineage+analysis%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">The spatial relationship between stem cells and their progeny in the basal layer of human epidermis: a new view based on whole-mount labelling and lineage analysis.</a> Development. 126 (11), 2409-2418 (1999).</li>
<li>Braun, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Manipulation+of+stem+cell+proliferation+and+lineage+commitment%3A+visualisation+of+label-retaining+cells+in+wholemounts+of+mouse+epidermis%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Manipulation of stem cell proliferation and lineage commitment: visualisation of label-retaining cells in wholemounts of mouse epidermis.</a> Development. 130 (21), 5241-5255 (2003).</li>
<li>Driskell, R. R., Giangreco, A., Jensen, K. B., Mulder, K. W., Watt, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sox2-positive+dermal+papilla+cells+specify+hair+follicle+type+in+mammalian+epidermis%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Sox2-positive dermal papilla cells specify hair follicle type in mammalian epidermis.</a> Development. 136 (16), 2815-2823 (2009).</li>
<li>Niculescu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conditional+ablation+of+integrin+alpha-6+in+mouse+epidermis+leads+to+skin+fragility+and+inflammation%5BTitle%5D)+AND+%22Eur+J+Cell+Biol%22%5BJournal%5D)">Conditional ablation of integrin alpha-6 in mouse epidermis leads to skin fragility and inflammation.</a> Eur J Cell Biol. 90 (2-3), 270-277 (2011).</li>
<li>Fujiwara, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+basement+membrane+of+hair+follicle+stem+cells+is+a+muscle+cell+niche%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">The basement membrane of hair follicle stem cells is a muscle cell niche.</a> Cell. 144 (4), 577-589 (2011).</li>
<li>Desciak, E. B., Maloney, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Artifacts+in+frozen+section+preparation%5BTitle%5D)+AND+%22Dermatol+Surg%22%5BJournal%5D)">Artifacts in frozen section preparation.</a> Dermatol Surg. 26 (5), 500-504 (2000).</li>
<li>Driskell, R., Jahoda, C. A., Chuong, C. M., Watt, F., Horsley, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Defining+dermal+adipose+tissue%5BTitle%5D)+AND+%22Exp+Dermatol%22%5BJournal%5D)">Defining dermal adipose tissue.</a> Exp Dermatol. 23 (9), 629-631 (2014).</li>
<li>Driskell, R. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distinct+fibroblast+lineages+determine+dermal+architecture+in+skin+development+and+repair%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Distinct fibroblast lineages determine dermal architecture in skin development and repair.</a> Nature. 504 (7479), 277-281 (2013).</li>
<li>Snippert, H. J., Schepers, A. G., Delconte, G., Siersema, P. D., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Slide+preparation+for+single-cell-resolution+imaging+of+fluorescent+proteins+in+their+three-dimensional+near-native+environment%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Slide preparation for single-cell-resolution imaging of fluorescent proteins in their three-dimensional near-native environment.</a> Nat Protoc. 6 (8), 1221-1228 (2011).</li>
<li>Sada, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Defining+the+cellular+lineage+hierarchy+in+the+interfollicular+epidermis+of+adult+skin%5BTitle%5D)+AND+%22Nat+Cell+Biol%22%5BJournal%5D)">Defining the cellular lineage hierarchy in the interfollicular epidermis of adult skin.</a> Nat Cell Biol. 18 (6), 619-631 (2016).</li>
<li>Chung, K., Deisseroth, K. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/23722210">CLARITY for mapping the nervous system.</a> Nat Methods. 10 (6), 508-513 (2013).</li>
<li>Tetteh, P. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Replacement+of+Lost+Lgr5-Positive+Stem+Cells+through+Plasticity+of+Their+Enterocyte-Lineage+Daughters%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Replacement of Lost Lgr5-Positive Stem Cells through Plasticity of Their Enterocyte-Lineage Daughters.</a> Cell Stem Cell. 18 (2), 203-213 (2016).</li>
</ol>

The authors would like to disclose that the publication of this manuscript was funded by Thermo-Fisher Scientific Inc.

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 &#181;m 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 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 &#181;m, 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 &#34;horizontal whole mount,&#34; 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 &#181;m-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&#8212;classical cryosectioning and thick, 3D tissue cross-sectioning&#8212;highlighting the advantages of &#34;horizontal whole mounts&#34; for the potential user of this protocol.

<table><tbody><tr><td>PBS</td><td></td><td></td><td>homemade</td></tr><tr><td>Gelatin, from cold water fish skin</td><td>Sigma</td><td>G7765</td><td>250ML</td></tr><tr><td>Glycerol</td><td>BDH Laboratory Supplies</td><td>444482V</td><td></td></tr><tr><td>O.C.T. compound</td><td>VWR chemicals</td><td>361603E</td><td></td></tr><tr><td>Peel-A-Way embedding molds</td><td>Sigma</td><td>E6032-1CS</td><td>Square S-22</td></tr><tr><td>Non-Fat Powdered Milk</td><td>Bio Basic Inc.</td><td>NB0669</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T9284</td><td>500ML</td></tr><tr><td>4&amp;prime;,6-Diamidino-2-phenylindole, dilactate (DAPI)</td><td>Invitrogen</td><td>D3571</td><td></td></tr><tr><td>FITC Rat Anti-Human CD49f</td><td>BD Pharmingen</td><td>555735</td><td></td></tr><tr><td>Mouse/Rat Integrin alpha 8 Antibody</td><td>R&amp;amp;D Systems</td><td>AF4076</td><td></td></tr><tr><td>Alexa Fluor 488 donkey anti-rat IgG</td><td>Life Technologies</td><td>A21208</td><td></td></tr><tr><td>Alexa Fluor 555 donkey anti-goat IgG</td><td>Life Technologies</td><td>A21432</td><td></td></tr><tr><td>CryoStar NX70</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>100mm Culture dishes</td><td></td><td></td><td></td></tr><tr><td>Disposable scalpels</td><td>Swann-Morton</td><td>Ref 0501</td><td></td></tr><tr><td>pointed metal forceps</td><td></td><td></td><td></td></tr><tr><td>1.5 ml microcentrifuge tubes</td><td>VWR</td><td>211-2130</td><td></td></tr><tr><td>12 Well plates</td><td>Sigma</td><td>CLS3513</td><td></td></tr><tr><td>Dissecting microscope</td><td>Nikon</td><td></td><td></td></tr><tr><td>20 ul and 1000 ul Pipette</td><td>Gilson</td><td></td><td></td></tr><tr><td>1000&amp;micro;l XL Graduated TipOne Filter Tip (Sterile)</td><td>Star Lab</td><td>S1122-1830</td><td></td></tr><tr><td>20&amp;micro;l Bevelled TipOne Filter Tip (Sterile)</td><td>Star Lab</td><td>S1120-1810</td><td></td></tr><tr><td>Rocking shaker plate</td><td></td><td></td><td></td></tr><tr><td>Microscope slides, menzel Glaeser</td><td>Thermos Scientific</td><td>631-9483</td><td>Superfrost Plus</td></tr><tr><td>Cover Glasses, Menzel Glaeser</td><td>Thermos Scientific</td><td>MENZBB024060AB</td><td>24 x 60 mm</td></tr><tr><td>Confocal microscope</td><td>Nikon</td><td></td><td></td></tr></tbody></table>

horizontal whole mount: a novel processing and imaging protocol for thick, three-dimensional tissue cross-sections of skin

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 &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-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.

To emphasize the advantages of our technique, we compared our thick, 3D tissue cross-sectioning technique, &#34;horizontal whole mount,&#34; 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 &#34;goosebumps&#34;), 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-&#181;m 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.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56106/56106fig4.jpg" /> 
Figure 4. Horizontal whole mount compared to a classical cryosection. 
(a) Classically obtained skin cryosections 10 &#181;m thick and (b) 100 &#181;m-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 &#181;m. The data is represented as Mean &#177; Standard Error of the Mean (SEM). One section per biological replicate (n=3) was quantified. Unpaired t-test *P&#60;0.05, ***P&#60;0.0005. <a href="http://cloudflare.jove.com/files/ftp_upload/56106/56106fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin at JoVE.com

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

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 ...

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Developmental Biology

15.1K Views.  King's College London. 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.

Video: Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

High-resolution Episcopic Microscopy (HREM) - Simple and Robust Protocols for Processing and Visualizing Organic Materials

We provide simple protocols for generating digital volume data with the high-resolution episcopic microscopy (HREM) method. HREM is capable of imaging organic materials with volumes up to 5 x 5 x 7 mm3 in typical numeric resolutions between 1 x 1 x 1 and 5 x 5 x 5 &#181;m3. Specimens are embedded in methacrylate resin and sectioned on a microtome. After each section an image of the block surface is captured with a digital video camera that sits on the phototube connected to the compound microscope head. The optical axis passes through a green fluorescent protein (GFP) filter cube and is aligned with a position, at which the bock holder arm comes to rest after each section. In this way, a series of inherently aligned digital images, displaying subsequent block surfaces are produced. Loading such an image series in three-dimensional (3D) visualization software facilitates the immediate conversion to digital volume data, which permit virtual sectioning in various orthogonal and oblique planes and the creation of volume and surface rendered computer models. We present three simple, tissue specific protocols for processing various groups of organic specimens, including mouse, chick, quail, frog and zebra fish embryos, human biopsy material, uncoated paper and skin replacement material.

High-resolution Episcopic Microscopy HREM - Simple and Robust Protocols for Processing and Visualizing Organic Materials

We provide simple and robust protocols for processing biopsy material of various species, embryos of biomedical model organisms and samples of other organic tissues in order to permit digital volume data generation with the high-resolution episcopic microscopy method.

We provide simple protocols for generating digital volume data with the high-resolution episcopic microscopy (HREM) method. HREM is capable of imaging ...

Intact Histological Characterization of Brain-implanted Microdevices and Surrounding Tissue

Research into the design and utilization of brain-implanted microdevices, such as microelectrode arrays, aims to produce clinically relevant devices that interface chronically with surrounding brain tissue. Tissue surrounding these implants is thought to react to the presence of the devices over time, which includes the formation of an insulating "glial scar" around the devices. However, histological analysis of these tissue changes is typically performed after explanting the device, in a process that can disrupt the morphology of the tissue of interest.
Here we demonstrate a protocol in which cortical-implanted devices are collected intact in surrounding rodent brain tissue. We describe how, once perfused with fixative, brains are removed and sliced in such a way as to avoid explanting devices. We outline fluorescent antibody labeling and optical clearing methods useful for producing an informative, yet thick tissue section. Finally, we demonstrate the mounting and imaging of these tissue sections in order to investigate the biological interface around brain-implanted devices.

Here we present a histological method for capturing, labeling, optically clearing, and imaging the intact brain tissue interface around chronically implanted microdevices in rodent brain tissue. Results from the techniques comprising this method are useful for understanding the impact of various penetrating brain-implants on their surrounding tissue.

Research  into the design and utilization of brain-implanted microdevices, such as  microelectrode arrays, aims to produce clinically relevant devices ...

Neuroscience

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

Here, we present a protocol of a whole-mount adult ear skin imaging technique to study comprehensive three-dimensional neuro-vascular branching morphogenesis and patterning, as well as immune cell distribution at a cellular level. The analysis of peripheral nerve and blood vessel anatomical structures in adult tissues provides some insights into the understanding of functional neuro-vascular wiring and neuro-vascular degeneration in pathological conditions such as wound healing. As a highly informative model system, we have focused our studies on adult ear skin, which is readily accessible for dissection. Our simple and reproducible protocol provides an accurate depiction of the cellular components in the entire skin, such as peripheral nerves (sensory axons, sympathetic axons, and Schwann cells), blood vessels (endothelial cells and vascular smooth muscle cells), and inflammatory cells. We believe this protocol will pave the way to investigate morphological abnormalities in peripheral nerves and blood vessels as well as the inflammation in the adult ear skin under different pathological conditions.

Here, we describe a high resolution whole-mount imaging method in the entire adult mouse ear skin, which enables us to visualize branching morphogenesis and patterning of peripheral nerves and blood vessels, as well as immune cell distribution.

Here, we present a protocol of a whole-mount adult ear skin imaging technique to study comprehensive three-dimensional neuro-vascular branching ...

Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel cell clusters), sebaceous glands, nerves and nerve endings, and capillaries. The spatial arrangement of these structures is tightly controlled on a microscopic scale - as seen, for example, in the orderly arrangement of cell types within a single hair follicle - and on a macroscopic scale - as seen by the nearly identical orientations of thousands of hair follicles within a local region of skin. Visualizing these structures without physically sectioning the skin is possible because of the 2-dimensional geometry of this organ. In this protocol, we show that mouse skin can be dissected, fixed, permeabilized, stained, and clarified as an intact two dimensional object, a flat mount. The protocol allows for easy visualization of skin structures in their entirety through the full thickness of large areas of skin by optical sectioning and reconstruction. Images of these structures can also be integrated with information about position and orientation relative to the body axes.

Mammalian skin contains a diverse array of structures - such as hair follicles and nerve endings - that exhibit distinctive patterns of spatial organization. Analyzing skin as a flat mount takes advantage of the 2-dimensional geometry of this tissue to produce full-thickness high-resolution images of skin structures.

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel ...

Three-Dimensional Imaging of Tumor-Bearing Tissue Using the Iterative Bleaching Extends Multiplexity Approach

The spatial heterogeneity of the tumor microenvironment (TME) is a critical determinant of therapeutic response, particularly for immune-oncology agents, where success depends on the distribution of specific immune cell subpopulations. Over the past decade, multiple sophisticated technologies have been introduced to achieve detailed resolution of the TME using two-dimensional sections from either formalin-fixed, paraffin-embedded (FFPE) or fixed-frozen tissues. While these thin sections are easier to procure and analyze, they lack the three-dimensional architecture needed to reliably and comprehensively characterize a tumor. To address this limitation, a tissue mounting and imaging technique was developed to enable the three-dimensional analysis of tumor lesions in their native in vivo state. This protocol outlines the procurement of human tumor tissue, the mounting of samples on custom-printed platforms, and staining procedures for post-fixation samples. The multiplexed immunofluorescence technique, IBEX (Iterative Bleaching Extends Multiplexity), was adapted to characterize the three-dimensional TME with up to 15 markers for tumor, immune, and stromal cells using commercially available antibodies. Imaging depths of up to 100 &#181;m were achieved using an inverted white-light laser confocal microscope with a custom-printed imaging adaptor and commercial glass-bottom dishes to ensure optimal tissue orientation. This protocol highlights the potential of the IBEX method to expand multiplexed immunofluorescence studies, providing a more comprehensive understanding of TME composition.

This article describes a protocol for multiplex immunofluorescence optimized to characterize the three-dimensional architecture of peritoneal metastases.

The spatial heterogeneity of the tumor microenvironment (TME) is a critical determinant of therapeutic response, particularly for immune-oncology agents, ...

Cancer Research

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Bioengineering

Generation of Self-assembled Vascularized Human Skin Equivalents

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to study skin development, wound healing, and grafting techniques. Many HSEs continue to lack vasculature and are additionally analyzed through post-culture histological sectioning which limits volumetric assessment of the structure. Presented here is a straightforward protocol utilizing accessible materials to generate vascularized human skin equivalents (VHSE); further described are volumetric imaging and quantification techniques of these constructs. Briefly, VHSEs are constructed in 12 well culture inserts in which dermal and epidermal cells are seeded into rat tail collagen type I gel. The dermal compartment is made up of fibroblast and endothelial cells dispersed throughout collagen gel. The epidermal compartment is made up of keratinocytes (skin epithelial cells) that differentiate at the air-liquid interface. Importantly, these methods are customizable based on needs of the researcher, with results demonstrating VHSE generation with two different fibroblast cell types: human dermal fibroblasts (hDF) and human lung fibroblasts (IMR90s). VHSEs were developed, imaged through confocal microscopy, and volumetrically analyzed using computational software at 4- and 8-week timepoints. An optimized process to fix, stain, image, and clear VHSEs for volumetric examination is described. This comprehensive model, imaging, and analysis techniques are readily customizable to the specific research needs of individual labs with or without prior HSE experience.

The goal of this protocol is to describe the generation and volumetric analysis of vascularized human skin equivalents using accessible and simple techniques for long term culture. To the extent possible, the rationale for steps is described to allow researchers the ability to customize based on their research needs.

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to ...

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods.
Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

Biology

Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can also be used to investigate and validate novel techniques and algorithms in volumetric medical imaging and therapies. Creating 3D high-resolution atlases of different organs1,2,3 is another application of histology volume reconstruction. This provides a resource for investigating tissue structures and the spatial relationship between various cellular features. We present an image registration approach for histology volume reconstruction, which uses a set of optical blockface images. The reconstructed histology volume represents a reliable shape of the processed specimen with no propagated post-processing registration error. The Hematoxylin and Eosin (H&#38;E) stained sections of two mouse mammary glands were registered to their corresponding blockface images using boundary points extracted from the edges of the specimen in histology and blockface images. The accuracy of the registration was visually evaluated. The alignment of the macrostructures of the mammary glands was also visually assessed at high resolution.
This study delineates the different steps of this image registration pipeline, ranging from excision of the mammary gland through to 3D histology volume reconstruction. While 2D histology images reveal the structural differences between pairs of sections, 3D histology volume provides the ability to visualize the differences in shape and volume of the mammary glands.

We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands.

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can ...

Whole&#45;Organ Pathogen Tracking via Fluorescent Reporter Quantification

High&#45;Resolution Three&#45;Dimensional Whole&#45;Organ Tomography of Microbial Infections

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen cells within this diverse environment significantly affects their stress levels, responses, fate, and contribution to the overall progression of the disease and treatment failure. However, due to the technical difficulties in locating &#181;m-sized pathogen cells within cm-sized host organs, this area of research has been relatively unexplored. Here, we present a method for addressing this challenge. We employ serial two-photon tomography and AI-enhanced image analysis to locate individual Salmonella cells throughout the entire spleen, liver lobes, and whole lymph nodes of infected mice. Using fluorescent reporters and in vivo antibody administration, the replication rate of single Salmonella cells, their local interaction with specific immune cells, and bacterial responses to antibiotics can be determined. These methodologies open avenues for a comprehensive examination of infections, their prevention, and treatment within the three-dimensional tissue context.

Author Spotlight: Unraveling Bacterial Responses to Antibiotics and Immune System in Tissues

Here, we describe a procedure that enables organ-wide detection of pathogenic bacteria during infection and quantification of fluorescent reporter activities.

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen ...