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

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.