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="https://cloudfront-jove-com.remotexs.ntu.edu.sg/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="https://cloudfront-jove-com.remotexs.ntu.edu.sg/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="https://cloudfront-jove-com.remotexs.ntu.edu.sg/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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+dermal+BMP+signalling+regulates+stem+cell+activation+during+hair+regeneration.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+self-renewal+and+differentiation+phases+in+the+niche+of+infrequently+dividing+hair+follicle+stem+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulation+of+stem+cell+proliferation+and+lineage+commitment:+visualisation+of+label-retaining+cells+in+wholemounts+of+mouse+epidermis.">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. 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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 border="0" cellpadding="0" cellspacing="0" width="658">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">PBS</td>
			<td style="width:99px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">homemade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Gelatin, from cold water fish skin</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:119px;">G7765</td>
			<td>250ML</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:273px;">Glycerol</td>
			<td style="width:99px;">BDH Laboratory Supplies</td>
			<td style="width:119px;">444482V</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">O.C.T. compound</td>
			<td style="width:99px;">VWR chemicals</td>
			<td style="width:119px;">361603E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Peel-A-Way embedding molds</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:119px;">E6032-1CS</td>
			<td>Square S-22</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Non-Fat Powdered Milk</td>
			<td style="width:99px;">Bio Basic Inc.</td>
			<td style="width:119px;">NB0669</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Triton X-100</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:119px;">T9284</td>
			<td>500ML</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">4&#8242;,6-Diamidino-2-phenylindole, dilactate (DAPI)</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:119px;">D3571</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">FITC Rat Anti-Human CD49f</td>
			<td style="width:99px;">BD Pharmingen</td>
			<td style="width:119px;">555735</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Mouse/Rat Integrin alpha 8 Antibody</td>
			<td style="width:99px;">R&#38;D Systems</td>
			<td style="width:119px;">AF4076</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Alexa Fluor 488 donkey anti-rat IgG</td>
			<td style="width:99px;">Life Technologies</td>
			<td style="width:119px;">A21208</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Alexa Fluor 555 donkey anti-goat IgG</td>
			<td style="width:99px;">Life Technologies</td>
			<td style="width:119px;">A21432</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:273px;">CryoStar NX70</td>
			<td style="width:99px;">Thermo Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">100mm Culture dishes</td>
			<td style="width:99px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Disposable scalpels</td>
			<td style="width:99px;">Swann-Morton</td>
			<td style="width:119px;">Ref 0501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">pointed metal forceps</td>
			<td style="width:99px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">1.5 ml microcentrifuge tubes</td>
			<td style="width:99px;">VWR</td>
			<td dir="LTR">211-2130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">12 Well plates</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:119px;">CLS3513</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Dissecting microscope</td>
			<td style="width:99px;">Nikon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">20 ul and 1000 ul Pipette</td>
			<td style="width:99px;">Gilson</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;">1000&#181;l XL Graduated TipOne Filter Tip (Sterile)</td>
			<td style="width:99px;">Star Lab</td>
			<td dir="LTR">S1122-1830</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;">20&#181;l Bevelled TipOne Filter Tip (Sterile)</td>
			<td style="width:99px;">Star Lab</td>
			<td>S1120-1810</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Rocking shaker plate</td>
			<td style="width:99px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Microscope slides, menzel Glaeser</td>
			<td style="width:99px;">Thermos Scientific</td>
			<td style="width:119px;">631-9483</td>
			<td>Superfrost Plus</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Cover Glasses, Menzel Glaeser</td>
			<td style="width:99px;">Thermos Scientific</td>
			<td style="width:119px;">MENZBB024060AB</td>
			<td>24 x 60 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Confocal microscope</td>
			<td style="width:99px;">Nikon</td>
			<td style="width:119px;"></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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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 ...

horizontal-whole-mount-novel-processing-imaging-protocol-for-thick

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

14.9K 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

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, especially, in plant reproductive biology. However, plant morphological, anatomical, and ultrastructural analyses traditionally involve time-consuming embedding and sectioning procedures for bright field, scanning, and electron microscopy. Recent progress in confocal fluorescence microscopy, state-of-the-art 3-D computer-aided microscopic analyses, and the continuous refinement of molecular techniques to be used on minimally processed whole-mount specimens, has led to an increased demand for developing efficient and minimal sample processing techniques. In this protocol, we describe techniques for properly dissecting Arabidopsis flowers and siliques, basic clearing techniques, and some staining procedures for whole-mount observations of reproductive structures.

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

In this protocol, we describe techniques for the proper dissection of Arabidopsis flowers and siliques, some basic clearing techniques, and selected staining procedures for whole-mount observations of reproductive structures.

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva

Periodontal diseases (such as gingivitis and periodontitis) are the leading causes of tooth loss in adults. Inflammation in gingiva is the fundamental physiopathology of periodontal diseases. Current experimental models of periodontal diseases have been established in various types of animals. However, the physiopathology of animal models is different from that of humans, making it difficult to analyze cellular and molecular mechanisms and evaluate new medicines for periodontal diseases. Here, we present a detailed protocol for reconstructing human inflammatory tissue equivalents of gingiva (iGTE) in vitro. We first build human tissue equivalents of gingiva (GTE) by utilizing two types of human cells, including human gingival fibroblasts (HGF) and human skin epidermal keratinocytes (HaCaT), under three-dimensional conditions. We create a wound model by using a tissue puncher to punch a hole in the GTE. Next, human THP-1 monocytes mixed with collagen gel are injected into the hole in the GTE. By adimistration of 10 ng/mL phorbol 12-myristate 13-acetate (PMA) for 72 h, THP-1 cells differentiated into macrophages to form inflammatory foci in GTE (iGTE) (IGTE also can be stumilated with 2 &#181;g/mL of lipopolysaccharides (LPS) for 48 h to initiate inflammation). IGTE is the first in vitro model of inflammatory gingiva using human cells with a three-dimensional architecture. IGTE reflects major pathological changes (immunocytes activition, intracellular interactions among fibryoblasts, epithelial cells, monocytes and macrophages) in periodontal diseases. GTE, wounded GTE, and iGTE can be used as versatile tools to study wound healing, tissue regeneration, inflammation, cell-cell interaction, and screen potential medicines for periodontal diseases.

The goal of the protocol is to build an inflammatory human gingiva model in vitro. This tissue model co-cultivates three types of human cells, HaCaT keratinocytes, gingival fibroblasts, and THP-1 macrophages, under three-dimensional conditions. This model can be applied to investigating periodontal diseases, such as gingivitis and periodontitis.

Periodontal diseases (such as gingivitis and periodontitis) are the leading causes of tooth loss in adults. Inflammation in gingiva is the fundamental ...

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 ...

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery.

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...

Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ovarian fitness is often assessed by visualizing and quantifying follicles and oocytes. Because the ovary is an opaque three-dimensional tissue, traditional approaches require laboriously slicing the tissue into numerous serial sections in order to visualize cells throughout the entire organ. Furthermore, because quantification by this method typically entails scoring only a subset of the sections separated by the approximate diameter of an oocyte, it is prone to inaccuracy. Here, a protocol is described that instead utilizes whole organ tissue clearing and immunofluorescence staining of mouse ovaries to visualize follicles and oocytes. Compared to more traditional approaches, this protocol is advantageous for visualizing cells within the ovary for numerous reasons: 1) the ovary remains intact throughout sample preparation and processing; 2) small ovaries, which are difficult to section, can be examined with ease; 3) cellular quantification is more readily and accurately achieved; and 4) the whole organ imaged.

Here, we present a protocol to quantify follicles in cultured ovaries of young mice without serial sectioning. Using whole organ immunofluorescence and tissue clearing, physical sectioning is replaced with optical sectioning. This method of sample preparation and visualization maintains organ integrity and facilitates automated quantification of specific cells.

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...