Name: detection and quantitation of label-retaining cells in mouse incisors using a 3d reconstruction approach after tissue clearing
Uploaded: 2022-06-10T14:00:00
Description: 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

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>
	...

<ol>
	<li>Seidel, K., 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=Resolving+stem+and+progenitor+cells+in+the+adult+mouse+incisor+through+gene+co-expression+analysis.">Resolving stem and progenitor cells in the adult mouse incisor through gene co-expression analysis.</a> eLife. 6, 24712 (2017).</li><li>Yu, T., Volponi, A. A., Babb, R., An, Z., Sharpe, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+in+tooth+development,+growth,+repair,+and+regeneration.">Stem cells in tooth development, growth, repair, and regeneration.</a> Current Topics in Developmental Biology. 115, 187-212 (2015).</li><li>An, Z., 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=A+quiescent+cell+population+replenishes+mesenchymal+stem+cells+to+drive+accelerated+growth+in+mouse+incisors.">A quiescent cell population replenishes mesenchymal stem cells to drive accelerated growth in mouse incisors.</a> Nature Communications. 9 (1), 378 (2018).</li><li>Biehs, B., 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=BMI1+represses+Ink4a/Arf+and+Hox+genes+to+regulate+stem+cells+in+the+rodent+incisor.">BMI1 represses Ink4a/Arf and Hox genes to regulate stem cells in the rodent incisor.</a> Nature Cell Biology. 15 (7), 846-852 (2013).</li><li>Sharir, A., 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=A+large+pool+of+actively+cycling+progenitors+orchestrates+self-renewal+and+injury+repair+of+an+ectodermal+appendage.">A large pool of actively cycling progenitors orchestrates self-renewal and injury repair of an ectodermal appendage.</a> Nature Cell Biology. 21 (9), 1102-1112 (2019).</li><li>Terskikh, V. V., Vasiliev, A. V., Vorotelyak, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label+retaining+cells+and+cutaneous+stem+cells.">Label retaining cells and cutaneous stem cells.</a> Stem Cell Reviews and Reports. 8 (2), 414-425 (2012).</li><li>de Miguel-Gomez, L., 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=Stem+cells+and+the+endometrium:+From+the+discovery+of+adult+stem+cells+to+pre-clinical+models.">Stem cells and the endometrium: From the discovery of adult stem cells to pre-clinical models.</a> Cells. 10 (3), 595 (2021).</li><li>Ishikawa, Y., Nakatomi, M., Ida-Yonemochi, H., Ohshima, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quiescent+adult+stem+cells+in+murine+teeth+are+regulated+by+Shh+signaling.">Quiescent adult stem cells in murine teeth are regulated by Shh signaling.</a> Cell and Tissue Research. 369 (3), 497-512 (2017).</li><li>Chehrehasa, F., Meedeniya, A. C., Dwyer, P., Abrahamsen, G., Mackay-Sim, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EdU,+a+new+thymidine+analogue+for+labelling+proliferating+cells+in+the+nervous+system.">EdU, a new thymidine analogue for labelling proliferating cells in the nervous system.</a> Journal of Neuroscience Methods. 177 (1), 122-130 (2009).</li><li>Solius, G. M., Maltsev, D. I., Belousov, V. V., Podgorny, O. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+nucleotide+analogue-based+techniques+for+tracking+dividing+stem+cells:+An+overview.">Recent advances in nucleotide analogue-based techniques for tracking dividing stem cells: An overview.</a> The Journal of Biological Chemistry. 297 (5), 101345 (2021).</li><li>Ning, H., Albersen, M., Lin, G., Lue, T. F., Lin, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+EdU+labeling+on+mesenchymal+stem+cells.">Effects of EdU labeling on mesenchymal stem cells.</a> Cytotherapy. 15 (1), 57-63 (2013).</li><li>Lin, G., 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=Labeling+and+tracking+of+mesenchymal+stromal+cells+with+EdU.">Labeling and tracking of mesenchymal stromal cells with EdU.</a> Cytotherapy. 11 (7), 864-873 (2009).</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>Salic, A., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+method+for+fast+and+sensitive+detection+of+DNA+synthesis+in+vivo.">A chemical method for fast and sensitive detection of DNA synthesis in vivo.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (7), 2415-2420 (2008).</li><li>Challen, G. A., Goodell, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promiscuous+expression+of+H2B-GFP+transgene+in+hematopoietic+stem+cells.">Promiscuous expression of H2B-GFP transgene in hematopoietic stem cells.</a> PLoS One. 3 (6), 2357 (2008).</li><li>Kopecky, B. J., Duncan, J. S., Elliott, K. L., Fritzsch, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+reconstructions+from+optical+sections+of+thick+mouse+inner+ears+using+confocal+microscopy.">Three-dimensional reconstructions from optical sections of thick mouse inner ears using confocal microscopy.</a> Journal of Microscopy. 248 (3), 292-298 (2012).</li><li>Tian, T., Yang, Z., Li, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+clearing+technique:+Recent+progress+and+biomedical+applications.">Tissue clearing technique: Recent progress and biomedical applications.</a> Journal of Anatomy. 238 (2), 489-507 (2021).</li><li>Ariel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+beginner's+guide+to+tissue+clearing.">A beginner's guide to tissue clearing.</a> The International Journal of Biochemistry &amp; Cell Biology. 84, 35-39 (2017).</li><li>Molbay, M., Kolabas, Z. I., Todorov, M. I., Ohn, T. L., Erturk, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guidebook+for+DISCO+tissue+clearing.">A guidebook for DISCO tissue clearing.</a> Molecular Systems Biology. 17 (3), 9807 (2021).</li><li>Jing, D., 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=Tissue+clearing+of+both+hard+and+soft+tissue+organs+with+the+PEGASOS+method.">Tissue clearing of both hard and soft tissue organs with the PEGASOS method.</a> Cell Research. 28 (8), 803-818 (2018).</li><li>Jing, D., 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=Tissue+clearing+and+its+application+to+bone+and+dental+tissues.">Tissue clearing and its application to bone and dental tissues.</a> Journal of Dental Research. 98 (6), 621-631 (2019).</li><li>Zhao, H., 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=Secretion+of+shh+by+a+neurovascular+bundle+niche+supports+mesenchymal+stem+cell+homeostasis+in+the+adult+mouse+incisor.">Secretion of shh by a neurovascular bundle niche supports mesenchymal stem cell homeostasis in the adult mouse incisor.</a> Cell Stem Cell. 14 (2), 160-173 (2014).</li><li>Fink, J., Andersson-Rolf, A., Koo, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+stem+cell+lineage+tracing+and+deep+tissue+imaging.">Adult stem cell lineage tracing and deep tissue imaging.</a> BMB Reports. 48 (12), 655-667 (2015).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5 M EDTA</td>
			<td>Sigma Aldrcih</td>
			<td>E9884</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#215; 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>CuSO4</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 ...

This protocol describes a method for in vivo and three-dimensional visualization and quantification of the label-retaining cells of mouse incisor stem cell niche. This technique is time-saving and user-friendly for new learners. The 3D images are acquired without damaging the tissue.The 3D quantitation makes the data more reliable and accurate. To begin, dissect the mandibles. Fix the samples in 4%paraformaldehyde, and keep them at room temperature overnight.After the incubation, remove the muscles from the mandibles. Immerse them in 0.5-molar ethylenediaminetetraacetic acid solution to decalcify for four days with a daily medium change on a 37-degree Celsius shaker. Decolorize the mandibles in 15 milliliters of 25%Quadrol solution on a 37-degree Celsius shaker for one day to remove the remaining blood heme.For whole mount 5-ethynyl-2-prime-deoxyuridine staining, prepare a fresh 5-ethynyl-2-prime-deoxyuridine labeling cocktail as mentioned in the text. Rinse the mandibles with a phosphate-buffered saline with Tween 20 or PBST for 30 minutes on a 37-degree Celsius shaker. Repeat rinsing the mandibles with phosphate-buffered saline thrice for three minutes each, followed by incubation in a freshly prepared 5-ethynyl-2-prime-deoxyuridine labeling cocktail on a shaker overnight.Repeat the phosphate-buffered saline wash three times, and perform serial de-lipidation by placing the mandibles in the solutions mentioned in the text for six hours each in 37-degree Celsius shaker. Dehydrate the mandibles in tert-butanol-polyethylene glycol, or tB-PEG, solution for two days at 37 degrees Celsius with the daily medium change. After two days, clear the mandible in benzyl benzoate-polyethylene glycol clearing medium containing 75%benzyl benzoate, 22%polyethylene glycol-methyl-ether methacrylate average 500 or PEG-MMA500, and 3%Quadrol to obtain refractive index matching and until transparency is achieved.Mount the tissue-cleared whole mandible on brand cavity slides in the benzyl benzoate-polyethylene glycol clearing medium, and cover it with glass cover slides. In the confocal imaging software, set the desired image acquisition parameters. In the panel for fluorescent excitation, activate the laser required to excite fluorophores optimally.In the panel for fluorescent detection, move the slider to select the wavelengths to be measured. 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, and place the mounted mandible on the microscope stage. Turn on the fluorescent lamps, and choose an appropriate magnification objective to image regions of interest.In the live scan mode, 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 two micrometers or as per your requirement.Acquire and save Z-stack image files in lif format. Use an imaging software file converter to convert the Z-stack image file to the native format file type. In the imaging software, click the Arena button, then choose Image.Select the original lif file, and choose the merged file. The images will be imported into the imaging software. Double-click the imported file to open the image data set.In the imaging software, visit the Display Adjustment panel. In the Display Adjustment panel, click on the name of the channel. It is usually labeled as red in default mode.In the Image Properties window, click on the Mapped Color tab. In the Color Tab File option, choose Fire Flow, and then click on OK.Select the display color to distinguish the background fluorescence and the positive fluorescence from the sample. On the upper left side of the screen, in the Scene Properties pane, click on the blue icon labeled Add New Surface.Unselect the Volume icon to remove most of the background fluorescence, resulting in the positive fluorescence distinctly visible during manual segmentation. Next, start the manual segmentation of the region of interest, or 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 based on the samples to contour the ROI easily.Next, ensure that the pointer menu in the right corner is in Select mode. Adjust the slice position to locate the ROI in the tissue. As mentioned earlier, most background fluorescence is already removed.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, followed by segmenting the region of interest slice by slice. After the ROI drawing is finished, click on Create Surface to get a 3D geometry of the ROI.Click on the Edit button in the manual surface creation wizard, and select Mask All. 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 zero. In the Scene Properties pane, deselect the Surface object, and select the Volume object. In the Display Adjustment, deselect the original Red channel, and select the Masked Red channel.And adjust the color intensities to get the desired 3D-rendered and positively labeled fluorescence ROI. Create a new spot object by clicking on the Spots icon to quantify the 3D-rendered label-retaining cells by creating 3D spots that comparably overlap with 3D-rendered label-retaining cells. Use the bottom arrows to move between steps in the spot creation wizard.Click on the Finish button, and select the Statistic button. Next, select the Overall tab to find the value for the total number of spots in the image. After 5-ethynyl-2-prime-deoxyuridine labeling and the polyethylene glycol-associated solvent system tissue-clearing process, the transparent mandible was obtained.The modified sample was compared to a normal mandible without a tissue-clearing process, and a transparent mandible with 5-ethynyl-2-prime-deoxyuridine-labeling was subjected to confocal imaging. The optical section of the incisor showed label-retaining cells in the stem cell niche of the incisor apex in the XY plane. The 3D image construction of an incisor apex showed 5-ethynyl-2-prime-deoxyuridine positive label-retaining quiescent stem cells in both the epithelial and mesenchymal stem cell niche.The 5-ethynyl-2-prime-deoxyuridine positive cells were transferred into spots for quantification that comparably overlapped with the mesenchymal label-retaining cells. The spots created only for mesenchymal label-retaining cells and only for epithelial label-retaining cells are visible in the image. Quantification of epithelial and mesenchymal label-retaining cells was also performed.Properties to processing and clearing are the most important steps and should be optimized according to the tissue's use to obtain satisfactory results. This method can also be modified to visualize S2VGFP-level LRCs and in cell lineage tracing to obtain details on progeny location and their quantitation or contributions.

Research

JoVE Journal

Developmental Biology

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

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 ...

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological ...

Tracing Gene Expression Through Detection of &#946;-galactosidase Activity in Whole Mouse Embryos

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Visualization and Analysis of Pharyngeal Arch Arteries using Whole-mount Immunohistochemistry and 3D Reconstruction

Improper formation or remodeling of the pharyngeal arch arteries (PAAs) 3, 4, and 6 contribute to some of the most severe forms of congenital heart ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...