We thank Meghann K. Holt for editing the manuscript. This study was supported by NIH/NIDCR grants DE026461 and DE028345 and the startup funding from the Texas A&#38;M School of Dentistry to Dr. Xiaofang Wang.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) for Texas A&#38;M University College of Dentistry.
1. Preparation of the EdU labeling cocktail
<ol>
	<li>Prepare the cocktail stock solutions as described in Table 1.</li>
	<li>Prepare the EdU labeling cocktail as described in Table 1.</li>
</ol>
2. Preparation of the mice and EdU solution
<ol>
	<li>Prepare the EdU solution. Dissolve EdU in DMSO at 20 mg/mL and store in a -20 &#176;C freezer. 
	CAUTION: EdU is a mutagen. Users should wear appropriate personal protective equipment (PPE) when handling this substance.</li>
	<li>Inject 5-day-old C57BL/6J mice with the prepared EdU solution intraperitoneally at 3 &#181;L/g body weight. Mice must receive the solution once a day for 7 consecutive days. Next, they undergo a chase period for 6 weeks before being harvested for analysis (Figure 1). 
	&#8203;CAUTION: Be careful to avoid needle pricking when injecting EdU into the mice. After injecting EdU for 7 consecutive days, place the mice in a new cage. After the mice are in their new home, dispose of the bedding in the old cage using appropriate biohazard rules.</li>
</ol>
3. Sample preparation by trans-cardiac perfusion (postnatal 53-day-old mice after the 6-week chase period)
NOTE: The mouse liver should turn pale after&#160;successful trans-cardiac perfusion.
<ol>
	<li>Anesthetize mice with an intraperitoneal injection. They need to be given a combination of xylazine and ketamine anesthetics (Xylazine 10-12.5 mg/kg; Ketamine, 80-100 mg/kg body weight).</li>
	<li>Wait for ~10 min until the mouse no longer responds to painful stimuli (such as paw pinch reflexes).</li>
	<li>Place the mice in a supine position stabilized on&#160;Styrofoam support with needles in each paw.</li>
	<li>Expose the chest cavity by opening up the overlying skin using tweezers and dissecting scissors.</li>
	<li>Cut open the diaphragm using sharp scissors. Take care not to pierce the heart.</li>
	<li>Cut through the ribcage, grabbing the base of the sternum with clamp scissors to expose the heart.</li>
	<li>Transfer the mouse to the perfusion stage near the circulation pump in a fume hood.</li>
	<li>Insert the 25 G needle (from the tubing filled with phosphate-buffered saline (PBS)/4% paraformaldehyde (PFA) solution) into the apex of the left ventricle. Take care to keep the tip of the needle in the lumen of the ventricle. 
	CAUTION: PFA is moderately toxic and a probable carcinogen. Use PPE to handle PFA and prepare the solution under a chemical fume hood. Waste PFA requires proper disposal per institution guidelines.</li>
	<li>Cut the right ventricle using a sharp scissor immediately after inserting the needle into the left ventricle. This step allows for blood to flow out of the mouse and drain into the collecting dish.</li>
	<li>Perfuse the samples with 50 mL of PBS solution at a flow rate of 7 mL/min.</li>
	<li>After PBS perfusion, switch the stopcock to allow for a flow of 20 mL of 4% PFA solution at the same flow rate of 5 mL/min.</li>
	<li>While the circulation pump is still on, remove the needle from the left ventricle. 
	&#8203;NOTE: The mouse is now fixed for tissue collection. To preserve the fluorescence, light exposure should be avoided whenever possible throughout the entire protocol. While the tissue is processing, the 50 mL conical tube with the tissue samples may be covered with aluminum foil.</li>
</ol>
4. Tissue clearing of the mandible using the PEGASOS technique
NOTE: A 15 mL or 50 mL conical tube as per the volume of tissues can be used to keep the samples ready for treatment in each step. Samples are processed at 37 &#176;C shakers (~100 rpm) from steps 4.2 to 4.7. Use polypropylene-based plastic containers that are resistant to organic solvents to avoid melting&#160;the plastic. Alternatively, glassware can be used.
CAUTION: The PEGASOS tissue clearing technique uses toxic solutions such as Quadrol, polyethylene glycol (PEG), benzyl benzoate (BB), MMA500, etc. Appropriate PPE is required to avoid potential exposures.
<ol>
	<li>Dissect mandibles and further fix samples in 4% PFA. Keep them at room temperature overnight.</li>
	<li>Remove the muscles from the mandibles and immerse them in 0.5 M EDTA solution (pH 8.0) to decalcify for 4 days. During this period, perform a daily medium change on a 37 &#176;C shaker. 
	NOTE: It is desirable to remove the muscles from the sample as much as possible. This precaution will simplify the imaging process and reduce the autofluorescence from the muscles.</li>
	<li>Decolorize the mandibles with a 25% Quadrol solution (v/v in H2O) on a 37 &#176;C shaker for one day to remove the remaining blood heme.</li>
	<li>Complete whole mount EdU staining.
	<ol>
		<li>Prepare a fresh EdU labeling cocktail according to Table 1. 
		NOTE: The EdU labeling cocktail needs to be made fresh each time with freshly prepared ascorbate solution added for obtaining optimum EdU labeling results.</li>
		<li>Rinse the mandibles with PBST for 30 min on a 37 &#176;C shaker.</li>
		<li>Rinse the mandibles with PBS three times for 3 min each time.</li>
		<li>Incubate the mandibles in a freshly made EdU labeling cocktail on a 37 &#176;C shaker overnight.</li>
		<li>Rinse the mandibles with PBS three times for 3 min each time.</li>
	</ol>
	</li>
	<li>Perform serial de-lipidation in each of the following solutions for 6 h on 37 &#176;C: 
	30%&#160;tert-butanol (tB) solution: 75% v/v H2O, 22% v/v tB, and 3% v/v Quadrol. 
	50%&#160;tB solution: 50% v/v H2O, 47% v/v tB, and 3% v/v Quadrol. 
	70%&#160;tB solution: 30% v/v H2O, 67% v/v tB, and 3% v/v Quadrol 
	NOTE: The de-lipidation step is critical. De-lipidation can be done between 4-6 h in 30% and 50% tB solution and for 1 day in 70% tB solution.</li>
	<li>Dehydrate the mandibles in tB-PEG solution for 2 days with the following daily medium change: 75% tB, 22% v/v polyethylene glycol methyl-ether methacrylate average MMA500, and 3% v/v Quadrol at 37 &#176;C.</li>
	<li>Clear the mandible in benzyl benzoate (BB)-PEG clearing medium to obtain refractive index matching: 75% v/v BB, 22% v/v PEG-MMA500, and 3% Quadrol until transparency is achieved. 
	NOTE: We have information on refractive index matching. The inorganic and organic components in tissues such as water, lipids, proteins, minerals, organelles, etc. have a different refractive index (RI) in the range of 1.33 to 1.55. This RI mismatch between the tissue components hinders the imaging process. Transparency can be achieved by eliminating RI mismatch within the tissue. Immersing the tissue-cleared samples in the clearing medium of BB-PEG is advised. This step will give the uniform internal RI of ~1.54 (called RI matching) to the whole specimen, easing the imaging process.</li>
	<li>Preserve the mandibles in the BB-PEG tissue clearing medium at room temperature for storage and imaging. 
	&#8203;NOTE: It is good to complete the imaging as soon as possible to avoid the gradual loss of fluorescent reporters. However, the PEGASOS method will keep fluorescence reporters well preserved even after several weeks of storage20,21. For long-term storage, the samples can also be stored at 4&#176;C.</li>
</ol>
5. Confocal imaging of the tissue-cleared mandible
<ol>
	<li>Mount the tissue cleared whole mandible on brand cavity slides in the BB-PEG clearing medium and cover with glass cover slides. Avoid any bubbles.</li>
	<li>Perform image acquisition with a suitable laser scanning confocal microscope. Select Acquire and set the image acquisition parameters at 512 x 512 pixel resolution and 400 Hz acquisition speed. These settings are found in the acquisition mode menu of the software that controls the microscope.</li>
	<li>In the panel for fluorescent excitation, activate the laser required (TRITC) to optimally excite fluorophores (the probe we used for LRCs visualization has fluorescent properties such as Cy3 with excitation at 548 nm and emission at 563 nm of laser wavelength).</li>
	<li>In the panel for fluorescent detection, move the slider to select the wavelengths that will be measured (for example, between 555 to 625 nm for a Cy3-like fluorophore).</li>
	<li>Place the mounted mandible on the microscope stage to visualize and find the sample using white light and a lower magnification objective (2-10x).</li>
	<li>Add a drop of immersion oil with a refractive index of 1.52 on top of the coverslip to match the refractive index of the glass coverslip and objective. 
	NOTE: Match the refractive index of objectives, imaging media, and tissue as closely as possible to minimize the introduction of optical distortions during image acquisition.</li>
	<li>Turn on the fluorescent lamps and choose an appropriate magnification objective to image regions of interest. We used a 20x objective lens with a&#160;numerical aperture of 0.9 with a working distance of 1.95 mm. A longer working distance lens may be required when imaging larger tissue samples in the confocal microscope. Alternatively, imaging can be done easily using a light-sheet microscope for larger tissue samples.</li>
	<li>On the confocal imaging software, press the Live button to start live-scan mode. To maximize the dynamic range of the images and avoid pixel saturation, adjust the gain and laser intensity for the channel selected.</li>
	<li>Identify the field of view to visualize the entire region of the mandible in the X and Y dimensions. Set the upper and lower Z-stage acquisition parameters that accurately cover the volume of the stem cell niche in the mice incisor. Use a Z-step size of 2 &#181;m or as per your requirements. The newer versions of confocal microscopes should be able to automatically acquire z-stacks of the various overlapping regions.</li>
	<li>Acquire and save Z-stack image files in .lif format. 
	&#8203;NOTE: The .lif flies can be converted into .tiff files and used for other 3D analysis software.</li>
</ol>
6. Image processing, 3D image reconstruction, and quantification of label-retaining cells by creating a surface, segmentation of the Region of Interest (ROI), masking the ROI, and creating spots
NOTE: We used Imaris (Bitplane 9.0.1) for image processing and 3D reconstruction, but similar image processing steps can be conducted using other software suites (e.g., ImageJ/Fiji, 3D slicer, Avizo, Arivis, Amira, etc.).
<ol>
	<li>In the imaging software, click the Arena button, then choose Image. Select the original .lif file and choose merged file, the images will be imported into the imaging software. 
	NOTE:&#160;Alternatively, use an imaging software file converter to convert Z stack image file to the native format file type for the software. For example: convert the .lif files into .ims files using the Imaris File Converter and open the .ims files in the imaging software. Converting data to the native file format of the software will allow rapid file conversion and minimize errors.</li>
	<li>Double click the imported file to open the image data set.</li>
	<li>Visit the Display Adjustment panel,click the name of the channel. It is usually labeled as Red in default mode.
	<ol>
		<li>In the Image Properties window, click on the Mapped Color tab. In the Color Table File option, choose Fire Flow, and then click on OK. The display color is normally chosen to distinguish the background fluorescence and the positive fluorescence from the sample.</li>
	</ol>
	</li>
	<li>On the upper left side of the screen in the Scene &#8211; Properties pane, click on the blue icon labeled Add New Surface. Unselect the Volume icon which will remove most of the background fluorescence and the positive fluorescence are distinctly visible (see Step 6.4.2).
	<ol>
		<li>Start the process of manual segmentation of region of interest (ROI) by clicking on the Create tab and selecting the Skip Automatic Creation, Edit Manually tab. In the manual surface creation wizard, click on the Contour tab to choose the orientation (XY, or YZ, or XZ) based on the samples to easily contour the ROI. Here, we choose XY orientation.</li>
		<li>Next, make sure the pointer menu in the right corner is in Select mode. Adjust the Slice Position to locate the ROI in the tissue. You will notice that most of the background fluorescence have been removed with distinct positive fluorescence as mentioned in Step 6.4. Click on the Draw button and draw a tentative region of interest. Select the Visibility option to none to avoid interference from other areas than ROI.</li>
		<li>Repeat step 6.4.2. slice by slice to draw the entire region of interest.</li>
		<li>After the ROI drawing is finished, click on Create Surface to get a 3D geometry of the ROI.</li>
		<li>Click on the Edit button in the manual surface creation wizard and select Mask All.</li>
		<li>In the popped-out Mask Channel window, select Channel 1 in the channel selection options. Afterward, select Duplicate Channel Before Applying the Mask. In the mask settings, select Constant Inside/Outside and set the voxel outside surface to 0.</li>
		<li>In the Scene-Properties pane, deselect the Surface Object and select the Volume Object. In the Display Adjustment, deselect the original &#34;Red&#8221; channel and select the Masked Red channel and adjust the color intensities to get the desired 3D-rendered and positively labeled fluorescence ROI.</li>
	</ol>
	</li>
	<li>Create a new spot object. Begin by clicking on the Add on&#160;Spots icon to quantify the 3D-rendered LRCs by creating 3D spots that comparably overlap with 3D-rendered LRCs. Use the bottom arrows to move between steps in the spot creation wizard.
	<ol>
		<li>In the spot creation wizard, there will be four sequential steps of instructions to create spots automatically with the software. Make sure the Segment Only a Region of Interest option is selected in the first step. Click on the Next icon.</li>
		<li>In the second step, there will be an &#34;XYZ 3D box&#34;. Adjust the XYZ box to cover the ROI. Click on the Next icon.</li>
		<li>In the third step, go to the Source Channel options. Choose the masked red channel; input the estimated XY spot diameter comparable to fit and overlap the sample fluorescence dots. Click on the Next icon.</li>
		<li>In the fourth step, add the &#34;Quality&#34; filter type and adjust the lower intensity threshold by moving the sliding window across the &#39;Quality&#39; histogram until the majority of identifiable LRCs are labeled. 
		NOTE: The segmentation and therefore statistical readouts (example: number of cells) very much depend on the intensity threshold values. Be careful to precisely set the appropriate threshold values.</li>
		<li>Click on the Finish button and select the Statistic button. Select the Overall tab to find the value for the total number of spots in the image.</li>
		<li>Export statistics by clicking on the Tab Display to File button and receive the results in an .xls file.</li>
		<li>Use a suitable diagram to present the quantification results.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Multiple doses of injections (BrdU, EdU) are usually used on growing neonatal mice to label proliferating cells as much as possible1,6,13. The chasing period is considered a critical step regarding the renewal rate of tissues6,13. The mouse incisor renews itself around every month. This trait allows researchers to set the chasing period to 4 weeks or longer<sup class="xr...

The continuously growing mouse incisor is an excellent model for studying adult stem cells1. The epithelial (labial and lingual cervical loop) and mesenchymal stem cells (between the labial and lingual cervical loop) that reside on the proximal side of the incisor differentiate into ameloblasts, odontoblasts, and dental pulp cells. This unique process provides a source of cells for compensation of tissue loss and turnover2. Though several stem cell markers such as Sox2, Gli1, Thy1/CD90, Bmi1, etc. have been identified in vivo for the subsets of adult stem cells in mouse incisors, they are inadequate in representing the stem cell populations when used alone1,3,4,5. Visualizing long-living quiescent cells by nucleotide analog DNA labeling and retention could provide unbiased detection for most subsets of adult stem cells6. Further, this approach is useful among many stem cell identification methods7 for understanding cell behavior and the homeostasis of dental stem cell populations3,8. While the dividing stem cells would lose their DNA labeling after a considerable chase, the putative quiescent stem cells retain their DNA label, deeming them label-retaining cells (LRCs)6. DNA labeling and retention by non-dividing stem cells will mark and locate the putative adult stem cells in their niches.
Over the past years, thymidine analog 5-bromo-2&#8242;-deoxyuridine (BrdU) labeling replaced the cumbersome, time-consuming, and high-resolution microscopy incompatible 3H-thymidine DNA labeling method for cell proliferation assays6,9. In recent years, the 5-ethynyl-2&#180;-deoxyuridine (EdU) labeling technique has been increasingly used over BrdU. This pattern emerged due to several reasons. First, the BrdU method is slow and labor-intensive. User conditions are variable, and it is unable to preserve the ultrastructure in specimens (due to DNA denaturation). Likewise, the BrdU method loses the antigenicity of cells, thus making it inefficient for downstream functional analyses and assays such as the co-localization experiments and in vivo stem cell transplantation3,7,9,10,11,12. BrdU is also a teratogen, which is not suitable for labeling LRCs in embryonic development6. Also, the BrdU method is inefficient when used in the whole-mount specimens. The disadvantages are low penetration of antibodies in the deep part of specimens or the requirement of a long antibody incubation period for deep penetration13. EdU labeling escapes the steps of denaturing specimens, thus preserving the ultrastructure. This feature is advantageous for downstream functional analyses such as co-localization experiments and stem cell transplantation11,12. Also, EdU labeling is highly sensitive and rapid; specimen penetration is high due to the use of rapidly absorbed and smaller-sized fluorescent azides for detecting EdU labels through a Cu(I)-catalyzed [3 + 2] cycloaddition reaction (&#34;click&#34; chemistry)14.
Another increasingly applied&#160;DNA labeling method is the use of engineered transgenic mice. These mice express histone 2B green fluorescent protein (H2B-GFP) controlled by a tetracycline-responsive regulator element5,14. After feeding mice with tetracycline chow/water for a 4-week to 4-month chasing period, the GFP fluorescence will diminish in cycling cells and only LRCs retain the fluorescence6. The advantage of this method is that the labeled LRCs can be isolated and remain viable for cell culture or downstream functional analyses6,7. Some studies reported inaccurate labeling of quiescent stem cells when chased for long-term use. This result was due to a leaky background expression from the H2B-GFP strain and not the appropriate tetracycline-regulated response15.
Moreover, most literature in the past used the LRCs detection mainly on sectioned slides, which are two dimensional and often erroneously biased in showing the accurate location and number of LRCs. The approach displayed incorrect angles for sections of complex tissue structures16. The other method was to obtain 3D images from serial sections and perform post-image reconstructions. These steps were inaccurate because of image distortion from variations in each serial section due to compressed or stretched sections, resulting in missing information16,17,18. The method was also laborious and time-consuming.
To facilitate the whole-mount imaging of LRCs, samples need to be made clear while the fluorescence be&#160;well maintained. Current tissue clearing techniques can be classified into three major categories: organic solvent-based tissue clearing techniques, aqueous reagent-based tissue clearing techniques, and hydrogel-based tissue clearing techniques17,19. The polyethylene glycol (PEG)-associated solvent system (PEGASOS) has been recently developed. This approach renders nearly all types of tissues transparent and preserves endogenous fluorescence, including hard tissues such as bone and teeth20. The PEGASOS method has advantages over other tissue clearing methods, especially in clearing tooth and bone materials. Most other methods could only partially clear hard tissues, have long processing times, or require costly reagents21. Also, the PEGASOS method can efficiently preserve endogenous fluorescence over other methods.
This literature led us to create a new method for cell study. We combined the LRCs detection advantages of EdU&#160;labeling with the most superior 3D whole-mount imaging of tissue-cleared specimens; samples were processed with&#160;advanced polyethylene glycol (PEG)-associated solvent system (PEGASOS) tissue clearing technique15. Hard tissue transparency enabled us to reconstruct the 3D signal of LRCs fluorescence in vivo without breaking the teeth or mandible, creating a more accurate way to visualize and quantify LRCs.
In this study, we provide an innovative guide to visualize LRCs in the mouse incisor. We made a 3D visual approach to determine the location and quantity of LRCs within the mouse incisor stem cell niche. This project used EdU labeling, PEGASOS tissue clearing techniques, and confocal microscopy. Our method of EdU labeling the LRCs on whole-mount tissue and the use of a cleared and transparent specimen overcomes both the limitations of traditional sectioned slides and other disadvantageous DNA labeling methods. Thus, our technique will be suitable for studies on stem cell homeostasis requiring LRCs detection, especially on hard tissues. The protocol can be equally advantageous to those focusing on stem cell homeostasis in other tissues and organs.

<table><tbody><tr><td>0.5 M EDTA</td><td>Sigma Aldrcih</td><td>E9884</td><td></td></tr><tr><td>20 &amp;times; Objective/NA 0.9</td><td>Leica</td><td>507702</td><td></td></tr><tr><td>50 mL Falcon Centrifuge Tubes</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>BD PrecisionGlide Needle</td><td>BD</td><td>REF 305111</td><td></td></tr><tr><td>Bezyl benzoate (BB)</td><td>Sigma Aldrcih</td><td>409529</td><td></td></tr><tr><td>Bitplane 9.0.1</td><td>Imaris</td><td></td><td></td></tr><tr><td>BRAND cavity slides</td><td>Millipore Sigma</td><td>BR475505</td><td></td></tr><tr><td>C57BL/6J mice</td><td>Jackson Laboratory</td><td>Strain #:000664</td><td></td></tr><tr><td>Circulation Pump</td><td>VWR</td><td>23609-170</td><td></td></tr><tr><td>CuSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma Aldrcih</td><td>451657</td><td></td></tr><tr><td>DMSO</td><td>Sigma Aldrcih</td><td>D8418</td><td></td></tr><tr><td>EdU</td><td>Carboynth</td><td>NE08701</td><td></td></tr><tr><td>Heparin</td><td>Miiilipore Sigma</td><td>H3149</td><td></td></tr><tr><td>Imaging System</td><td>Olympus</td><td>DP27</td><td></td></tr><tr><td>LAS X Software</td><td>Leica</td><td></td><td></td></tr><tr><td>Olympus Stereo Microscope</td><td>Olympus</td><td>SZX16</td><td></td></tr><tr><td>Paraformaldehye</td><td>Sigma Aldrich</td><td>P6148</td><td></td></tr><tr><td>PBS</td><td>Sigma Aldrich</td><td>P4417</td><td></td></tr><tr><td>PEGMMA500</td><td>Sigma Aldrich</td><td>447943</td><td></td></tr><tr><td>Quadrol</td><td>Sigma Aldrich</td><td>122262</td><td></td></tr><tr><td>Sodium Ascorbate</td><td>Sigma Aldrich</td><td>11140</td><td></td></tr><tr><td>Sulfa-Cyanine 3 Azide</td><td>Lumiprobe</td><td>D1330</td><td></td></tr><tr><td>TBS-10X</td><td>Cell Signaling Technology</td><td>12498</td><td></td></tr><tr><td>TCS SP8 Confocal Microscope</td><td>Leica</td><td></td><td></td></tr><tr><td>tert-butanol (tB)</td><td>Sigma Aldrich</td><td>360538</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>X100</td><td></td></tr></tbody></table>

detection and quantitation of label-retaining cells in mouse incisors using a 3d reconstruction approach after tissue clearing

The murine incisor is an organ that grows continuously throughout the lifespan of the mouse. The epithelial and mesenchymal stem cells residing in the proximal tissues of incisors give rise to progeny that will differentiate into ameloblasts, odontoblasts, and pulp fibroblasts. These cells are crucial in supporting the sustained turnover of incisor tissues, making the murine incisor an excellent model for studying the homeostasis of adult stem cells. Stem cells are believed to contain long-living quiescent cells that can be labeled by nucleotide analogs such as 5-ethynyl-2&#180;-deoxyuridine (EdU). The cells retain this label over time and are accordingly named&#160;label-retaining cells (LRCs). Approaches for visualizing LRCs in vivo provide a robust tool for monitoring stem cell homeostasis. In this study, we described a method for visualizing and analyzing LRCs. Our innovative approach features LRCs in mouse incisors after tissue clearing and whole-mount EdU staining followed by confocal microscopy and a 3-dimensional (3D) reconstruction with the imaging software. This method enables 3D imaging acquisition and non-biased quantitation compared to traditional LRCs analysis on sectioned slides.

After EdU labeling and the PEGASOS tissue clearing process (Figure 2), the transparent mandible was obtained as shown in our image (Figure 3B). We compared the modified sample to a normal mandible without a tissue clearing process (Figure 3A). The transparent mandible (Figure 3B) with EdU labeling was subjected to confocal imaging. We focused on the incisor apex showing the stem cell niche as shown in (...

Watch this Scientific Journal Video about Detection and Quantitation of Label-Retaining Cells in Mouse Incisors using a 3D Reconstruction Approach after Tissue Clearing at JoVE.com

Detection and Quantitation of Label-Retaining Cells in Mouse Incisors using a 3D Reconstruction Approach after Tissue Clearing

The mouse incisor contains valuable label-retaining cells in its stem cell niche. We have a novel way to unbiasedly detect and quantify the label-retaining cells; our study used EdU labeling and a 3D reconstruction approach after PEGASOS tissue clearing of the mandible.

The murine incisor is an organ that grows continuously throughout the lifespan of the mouse. The epithelial and mesenchymal stem cells residing in the ...

detection-quantitation-label-retaining-cells-mouse-incisors-using-3d

Research

JoVE Journal

Developmental Biology

1.9K Views.  Texas A&M School of Dentistry. The mouse incisor contains valuable label-retaining cells in its stem cell niche. We have a novel way to unbiasedly detect and quantify the label-retaining cells; our study used EdU labeling and a 3D reconstruction approach after PEGASOS tissue clearing of the mandible.

Video: Detection and Quantitation of Label-Retaining Cells in Mouse Incisors using a 3D Reconstruction Approach after Tissue Clearing

Clonal Genetic Tracing using the Confetti Mouse to Study Mineralized Tissues

Labeling an individual cell in the body to monitor which cell types it can give rise to and track its migration through the organism or determine its longevity can be a powerful way to reveal mechanisms of tissue development and maintenance. One of the most important tools currently available to monitor cells in vivo is the Confetti mouse model. The Confetti model can be used to genetically label individual cells in living mice with various fluorescent proteins in a cell type-specific manner and monitor their fate, as well as the fate of their progeny over time, in a process called clonal genetic tracing or clonal lineage tracing. This model was generated almost a decade ago and has contributed to an improved understanding of many biological processes, particularly related to stem cell biology, development, and renewal of adult tissues. However, preserving the fluorescent signal until image collection and simultaneous capturing of various fluorescent signals is technically challenging, particularly for mineralized tissue. This publication describes a step-by-step protocol for using the Confetti model to analyze growth plate cartilage that can be applied to any mineralized or nonmineralized tissue.

This method describes the use of the R26R-Confetti (Confetti) mouse model to study mineralized tissues, covering all steps from the breeding strategy to the image acquirements. Included is a general protocol that can be applied to all soft tissues and a modified protocol that can be applied to mineralized tissues.

Labeling an individual cell in the body to monitor which cell types it can give rise to and track its migration through the organism or determine its ...

Rapid Isolation of Single Cells from Mouse and Human Teeth

Mouse and human teeth represent challenging organs for quick and efficient cell isolation for single-cell transcriptomic or other applications. The dental pulp tissue, rich in the extracellular matrix, requires a long and tedious dissociation process that is typically beyond the reasonable time for single-cell transcriptomics. For avoiding artificial changes in gene expression, the time elapsed from euthanizing an animal until the analysis of single cells needs to be minimized. This work presents a fast protocol enabling to obtain single-cell suspension from mouse and human teeth in an excellent quality suitable for scRNA-seq (single-cell RNA-sequencing). This protocol is based on accelerated tissue isolation steps, enzymatic digestion, and subsequent preparation of final single-cell suspension. This enables fast and gentle processing of tissues and allows using more animal or human samples for obtaining cell suspensions with high viability and minimal transcriptional changes. It is anticipated that this protocol might guide researchers interested in performing the scRNA-seq not only on the mouse or human teeth but also on other extracellular matrix-rich tissues, including cartilage, dense connective tissue, and dermis.

The current protocol presents a fast, efficient, and gentle method for isolating single cells suitable for single-cell RNA-seq analysis from a continuously growing mouse incisor, mouse molar, and human teeth.

Mouse and human teeth represent challenging organs for quick and efficient cell isolation for single-cell transcriptomic or other applications. The dental ...

Live Tracking of Dental Cells via Two-Photon Microscopy

Using Ex Vivo Live Imaging to Investigate Cell Divisions and Movements During Mouse Dental Renewal

The continuously growing mouse incisor is emerging as a highly tractable model system to investigate the regulation of adult epithelial and mesenchymal stem cells and tooth regeneration. These progenitor populations actively divide, move, and differentiate to maintain tissue homeostasis and regenerate lost cells in a responsive manner. However, traditional analyses using fixed tissue sections could not capture the dynamic processes of cellular movements and interactions, limiting our ability to study their regulations. This paper describes a protocol to maintain whole mouse incisors in an explant culture system and live-track dental epithelial cells using multiphoton timelapse microscopy. This technique adds to our existing toolbox for dental research and allows investigators to acquire spatiotemporal information on cell behaviors and organizations in a living tissue. We anticipate that this methodology will help researchers further explore mechanisms that control the dynamic cellular processes taking place during both dental renewal and regeneration.

Author Spotlight: Understanding Dynamic Cellular Behaviors in Adult Mouse Dental Tissue Renewal and Repairment 

Ex vivo live imaging is a powerful technique for studying the dynamic processes of cellular movements and interactions in living tissues. Here, we present a protocol that implements two-photon microscopy to live track dental epithelial cells in cultured whole adult mouse incisors.

The continuously growing mouse incisor is emerging as a highly tractable model system to investigate the regulation of adult epithelial and mesenchymal ...

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical ...

Neuroscience

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ tissues provides pivotal data on disease development and progression. Using conventional microscopy methods, immunolabeling is mostly restricted to thin sections obtained from paraffin-embedded or frozen samples. However, the limited 2D image plane of these thin sections may lead to the loss of crucial information on the complex structure of an infected organ and the cellular context of the infection. Modern multicolor, immunostaining-compatible tissue clearing techniques now provide a relatively fast and inexpensive way to study high-volume 3D image stacks of virus-infected organ tissue. By exposing the tissue to organic solvents, it becomes optically transparent. This matches the sample&#8217;s refractive indices and eventually leads to a significant reduction of light scattering. Thus, in combination with long free working distance objectives, large tissue sections up to 1 mm in size can be imaged by conventional confocal laser scanning microscopy (CLSM) at high resolution. Here, we describe a protocol to apply deep-tissue imaging after tissue clearing to visualize rabies virus distribution in infected brains in order to study topics like virus pathogenesis, spread, tropism, and neuroinvasion.

Novel, immunostaining-compatible tissue clearing techniques like the ultimate 3D imaging of solvent-cleared organs allow the 3D visualization of rabies virus brain infection and its complex cellular environment. Thick, antibody-labeled brain tissue slices are made optically transparent to increase imaging depth and to enable 3D analysis by confocal laser scanning microscopy.

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Biology

Imaging and Quantification of Intact Neuronal Dendrites via CLARITY Tissue Clearing

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the morphology of processes and substructures within these neurons. As such, much of what is known about brain function has arisen alongside developments in imaging technologies that allow further insight into how neurons are organized and connected in the brain. Improvements in tissue clearing have allowed for high-resolution imaging of thick brain slices, facilitating morphological reconstruction and analyses of neuronal substructures, such as dendritic arbors and spines. In tandem, advances in image processing software provide methods of quickly analyzing large imaging datasets. This work presents a relatively rapid method of processing, visualizing, and analyzing thick slices of labeled neural tissue at high-resolution using CLARITY tissue clearing, confocal microscopy, and image analysis. This protocol will facilitate efforts toward understanding the connectivity patterns and neuronal morphologies that characterize healthy brains, and the changes in these characteristics that arise in diseased brain states.

Neuronal dendritic morphology often underlies function. Indeed, many disease processes that affect the development of neurons manifest with a morphological phenotype. This protocol describes a simple and powerful method for analyzing intact dendritic arbors and their associated spines.

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the ...

Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by crossing the &#39;Tet-On&#39; system oTet-Cre mouse with a novel reverse tetracycline transactivator (rtTA) transgene linked to the Tert promoter, which we have demonstrated marks a novel population of adult brain stem cells. Here, administration of the tetracycline derivative doxycycline to mTert-rtTA::oTet-Cre mice will indelibly mark a population of cells that express a 4.4 kb fragment of the promoter region of the gene Tert. When combined the Rosa-mTmG reporter, mTert-rtTA::oTet-Cre::Rosa-mTmG mice will express membrane tdTomato (mTomato) until doxycycline treatment induces the replacement of mTomato expression with membrane EGFP (mGFP) in cells that also express Tert. Therefore, when these triple-transgenic lineage tracing mice receive doxycycline (the &#34;pulse&#34; period during which TERT expressing cells are marked), these cells will become indelibly marked mGFP+ cells, which can be tracked for any desirable amount of time after doxycycline removal (the &#34;chase&#34; period), even if Tert expression is subsequently lost. Brains are then perfusion-fixed and processed for immunofluorescence and other downstream applications in order to interpret changes to stem cell activation, proliferation, lineage commitment, migration to various brain niches, and differentiation to mature cell types. Using this system, any rtTA mouse can be mated to oTet-Cre and a Rosa reporter to conduct doxycycline-inducible &#34;pulse-chase&#34; lineage&#160;tracing experiments using markers of stem cells.

The ability to permanently mark stem cells and their progeny with a fluorophore using an inducible transgenic lineage tracing mouse line allows for spatial and temporal analysis of activation, proliferation, migration, and/or differentiation in vivo. Lineage tracing can reveal novel information about lineage commitment, response to intervention(s), and multipotency.

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by ...

3D Imaging of PDL Collagen Fibers during Orthodontic Tooth Movement in Mandibular Murine Model

Orthodontic tooth movement is a complex biological process of altered soft and hard tissue remodeling as a result of external forces. In order to understand these complex remodeling processes, it is critical to study the tooth and periodontal tissues within their 3D context and therefore minimize any sectioning and tissue artefacts. Mouse models are often utilized in developmental and structural biology, as well as in biomechanics due to their small size, high metabolic rate, genetics and ease of handling. In principle this also makes them excellent models for dental related studies. However, a major impediment is their small tooth size, the molars in particular. This paper is aimed at providing a step by step protocol for generating orthodontic tooth movement and two methods for 3D imaging of the periodontal ligament fibrous component of a mouse mandibular molar. The first method presented is based on a micro-CT setup enabling phase enhancement&#160;imaging of fresh collagen tissues. The second method is a bone clearing method using ethyl cinnamate that enables imaging through the bone without sectioning and preserves endogenous fluorescence. Combining this clearing method with reporter mice like Flk1-Cre;TdTomato provided a first of its kind opportunity to image the 3D vasculature in the PDL and alveolar bone.

We present a protocol for generating orthodontic tooth movement in mice and methods for 3D visualization of the collagen fibers and blood vessels of periodontal ligament without sectioning.

Orthodontic tooth movement is a complex biological process of altered soft and hard tissue remodeling as a result of external forces. In order to ...

Medicine

Imaging the Gut with "CLARITY"

CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging compatible Tissue hYdrogel) has recently evolved as a valuable technique involving acrylamide embedding to delipidate tissue (without sectioning) and to preserve the 3-D tissue structure for immunostaining. The technique is highly relevant in imaging the dynamic gut environment where different cell types interact during homeostasis and disease states. This method optimized for the mouse gut is described here, which helps to trace cell types like epithelia, enteroendocrine, neurons, glia, and the neuronal projections into the epithelia or enteroendocrine cells that mediate microbial sensing or nutrient chemo sensing respectively. The gut tissue (1-1.5 cm) is fixed in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) at 4 &#176;C overnight on day 1. On day 2, PFA is discarded, and the tissue is washed thrice with PBS. The tissue is hydrogel embedded to preserve its integrity by incubation in 4% hydrogel (acrylamide) solution in PBS (diluted from 30% ProtoGel) overnight at 4 &#176;C. On day 3, the tissue-hydrogel solution is incubated at 37&#160;&#176;C for 1 h to allow hydrogel polymerization. Tissue is then washed thrice gently with PBS to remove excess hydrogel. The subsequent step of delipidation (clearing) involves tissue incubation in sodium dodecyl sulfate (8% SDS in PBS) at 37 &#176;C for 2 days (days 4 &#38; 5) on a shaker at room temperature (RT). On day 6, the cleared tissue is thoroughly washed with PBS to remove SDS. Tissue can be immunostained by incubation in primary antibodies (diluted in 0.5% normal donkey serum in PBS containing 0.3% Triton X-100), overnight at 4&#176;C, and subsequent incubation in appropriate secondary Alexa Fluor antibodies for 1.5 h at RT, and nuclear staining with DAPI (1: 10000). The tissue is transferred onto a clean glass slide and mounted using VectaShield for confocal imaging.

We describe the protocol for passive CLARITY (PACT) staining of mouse intestine to enable visualization of subepithelial tissues, including neurons, glia and enteroendocrine cells (EEC) without tissue sectioning. The protocol involves hydrogel embedding of formaldehyde-fixed tissue, and subsequent delipidation using an anionic detergent to "clear" the tissue.

CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging compatible Tissue hYdrogel) has recently evolved as a valuable technique involving ...