Clonal Genetic Tracing using the Confetti Mouse to Study Mineralized Tissues

All mouse work was approved by the Ethical Committee on Animal Experiments (Stockholm North Committee/Norra Djurförsöksetiska Nämd) and conducted in accordance with The Swedish Animal Agency´s Provisions and Guidelines for Animal Experimentation.

1. Mouse breeding

1. To generate the strain, cross the R26R-Confetti mouse with the Cre line of interest. Wean the pups at 21 days of age.
   NOTE: Mice can be used for breeding from approximately 8 weeks of age, or according to the local guidelines. Genotyping (not described here) can be performed from biopsies collected at weaning. The age at weaning can be adjusted as per local guidelines.

2. Confetti labeling

NOTE: This labeling strategy is appropriate for tamoxifen inducible Cre strains.

1. Prepare a solution of up to 2.5 mg/mL tamoxifen in corn oil in a glass bottle. Dissolve by heating to 37 °C in an oven for up to 60 min, agitating every 5 min using a vortex until the powder has dissolved.
   NOTE: For increased doses of tamoxifen, solutions up to 20 mg/mL can be prepared using this method.
2. Store at 4 °C for up to 3 months, protected from light.
3. Induce recombination by administering 50 µL of 2.5 mg/mL tamoxifen dissolved in corn oil intraperitoneally on the first postnatal day (P1).
   NOTE: In principle, tamoxifen can be administered at any age, as early as the F1 generation. The dose of tamoxifen should be varied based on Cre expression, mouse age, and experimental application. Further information can be found in the discussion section. Cre negative mice treated in the same way can be used as a negative control for Confetti fluorescent signals.
   CAUTION: Ensure animal handlers are warned of the tamoxifen use and ensure that cage materials are disposed of according to local environmental and safety guidelines.

3. Tissue collection

1. Euthanize mice on postnatal day 27 (P27) by gradually filling the space containing the mice with carbon dioxide up to at least 80% by volume and maintaining this concentration for 3 min. Perform cervical dislocation. Keep mice on ice during the dissection of the other animals.
2. Dissect out the tissue of interest. Outlined below is a protocol for collecting the proximal tibial and distal femoral growth plates and articular cartilage of the knee, suitable for postnatal mice up to the age of 6 months.
   1. Remove the skin around the hind limbs as far as the foot, using sharp scissors and toothed forceps.
   2. Roughly trim the muscle and fat tissue from the legs with sharp scissors.
      NOTE: Do not clean the bones perfectly, because the surrounding tissues provide some structural support when sectioning, but ensure all skin is removed.
   3. Using a scalpel, cut through the soft tissue where the top of the leg meets the body until the femoral head is visible, then cut the ligaments in the hip joint to remove the leg.
      NOTE: Alternatively, if it is not possible to find the femoral head, cut through the femur close to the hip with strong scissors.
   4. Dissect the foot from the rest of the leg using a scalpel or sharp scissors.

4. Tissue fixation and processing

NOTE: Based on the tissue of interest, follow one of the two protocols detailed below (4.1 for soft tissues or 4.2 for mineralized mouse tissues over the age of approximately 45 days, as detailed below). For both methods, store the dissected samples in the 3.7% formaldehyde/phosphate buffered saline (PBS) solution on ice while dissecting the remaining animals.

1. For soft tissues and mineralized tibiae and femora up to the age of approximately P45, fix the tissue in precooled 3.7% formaldehyde/PBS for 6 h, rolling gently on a rotator at 4 °C. Use a volume at least 10x greater than that of the tissue. Continue directly to step 4.3.
2. For mineralized tissues, including the tibiae and femora over the age of approximately P45, a decalcification step is required.
   1. Prepare 1 L of 10% EDTA/dH₂O solution with the pH adjusted to 8.05 using sodium hydroxide. Next, dilute the formaldehyde to 3.7% using this solution. The working concentration of EDTA will be 9%.
   2. Fix the tissue by placing into precooled 3.7% formaldehyde/PBS overnight, gently rolling on a rotator at 4 °C.
   3. Place the tissue into precooled 3.7% formaldehyde/9% EDTA/dH₂O solution (pH 8.05), and gently rotate at 4 °C in a volume at least 10x greater than that of the tissue. Repeat this step by placing the bones into fresh, precooled 3.7% formaldehyde/9% EDTA/dH₂O for 3x over the next 48 h.
      NOTE: This short decalcification is enough to allow for sectioning of the femur and tibia bones of up to 6 months-old mice. For more densely mineralized tissues further decalcification may be required to improve histology.
3. Transfer the tissue into precooled 30% sucrose/dH₂O. Make sure to fill the container to the brim, and gently rotate overnight at 4 °C. Use a volume at least 10x greater than that of the tissue. The tissue will often float when first placed into this solution and sink to the bottom of the bottle by the morning.
4. To remove the residual sucrose solution, briefly wash the tissue by removing it from the sucrose solution with forceps and completely covering the sample with approximately 5 ml of optimal cutting temperature compound (OCT) for several seconds.
5. Fill a prelabeled cryomold with OCT and position the tissue at the bottom. Work quickly to avoid the samples sitting at room temperature for too long.
   NOTE: It is important to place the sample in a convenient orientation for the sectioning step. To increase the chances of sectioning through entire growth plate columns after tracing with Col2CreERT:Confetti, place the medial side facing down using the femoral head as a guide, and position the sample as flat to the bottom of the mold as possible.
6. Embed the sample by placing the mold onto dry ice and waiting for the OCT to entirely solidify. Place the cryomolds as flat as possible to avoid movement/sliding of the tissue in the mold. Once finished, store the samples at -20 °C, if they are not used immediately.
   NOTE: Use within 12 weeks, because with age the blocks become more difficult to section.

5. Sectioning

NOTE: Perform cryosectioning using a cryostat with disposable blades suitable for hard tissue. Any standard cryostat is suitable.

1. Remove the block from the mold and secure it to the chuck by applying OCT between the top of the block and the chuck and cooling in the cryostat at -20 °C for at least 5 min, so that the OCT has completely solidified.
2. Precool both the sample holder and the blade holder to -20 °C and then place the sample/chuck in the chuck holder and the blade in the blade holder to equilibrate for several min.
   NOTE: The specified temperatures may be varied by several degrees depending on the cryostat and the tissue of interest.
3. Use the cryostat to trim the block and cut tissue sections of a suitable thickness to allow for the visualization of the clones in the tissue of interest.
   1. For postnatal growth plate analysis, prepare sections of 30−160 µm and collect them on slides. Some cryostat models may only allow the collection of thick sections using the trim setting.
4. Air dry the slides at room temperature until they are completely dry.
   NOTE: The duration of this step can vary based on tissue properties and section thickness. The temperature of the room is also likely to affect the speed of this step.
5. Store the slides at -20 °C for up to 36 months, or if processing immediately, proceed to step 6.2.

6. Slide preparation and mounting

1. Remove the slide from the freezer and bring up to room temperature horizontally in a slide holder. Allow any condensation to evaporate.
2. Remove the OCT by using a Pasteur pipette to gently apply PBS at room temperature onto the slide. The thicker the section, the longer time is required. For 160 µm thick sections, perform two rinses: incubate once for 15 min and remove the liquid, and then apply fresh PBS for 5 min and remove the liquid.
   NOTE: This step is to remove the OCT and can be varied based on tissue properties and section thickness, as appropriate. The temperature of the room is likely to affect the speed of this step.
3. Mount the slides in a solution of 75% 2,2-thiodiethanol/dH₂O at room temperature with 60 mm long cover slips.
   NOTE: Slides can be prepared immediately before imaging, but the fluorescent signal is stable for 1−2 weeks if kept out of the light in a humidified chamber at 4 °C.

7. Imaging by confocal microscopy

1. Set up the microscope.
   1. Turn on the microscope and open the software. Click the Start System button.
   2. Select the channels by first clicking Smart Setup under the Acquisition tab and then selecting the three channels corresponding to red fluorescent protein (RFP), yellow fluorescent protein (YFP), and cyan fluorescent protein (CFP) under the Dye heading. Click Best signal then Apply to select the lasers.
      NOTE: The contents of the Acquisition tab are shown in Supplementary file 1A. If the required channels are not present, add them using the ‘+’ button under the Dye heading. The necessary information regarding laser, excitation wavelengths, and the range of recorded emission wavelengths is shown for the three Confetti fluorescent proteins in Supplementary file 1B-D.
   3. Select the 20x objective lens under the Objective heading in the Acquisition Mode tab.
2. Locate the growth plate or other tissue of interest.
   1. To provide a distinct view, first select the RFP channel by clicking its name in the Channels tab. This causes details of the RFP channel to be displayed in the Light Path tab. Click the tick-box beside T-PMT.
      NOTE: T-PMT channel shows nonreflected laser light, which is used to visualize the nonfluorescent parts of the tissue.
   2. Place the slide in the slide holder and position the growth plate or other tissue of interest directly between the light path and the objective lens.
   3. In order to simplify localizing the tissue, deselect the YFP and CFP channels using the tick-boxes under the Tracks heading of the Channels tab.
   4. Select the T-PMT channel by clicking its name in the Tracks heading. Click Live in the Acquisition tab and increase the Gain (Master) for the T-PMT channel in order to visualize the tissue section on the screen, which is shown in greyscale.
   5. Move the position of the slide using the joystick to locate the region of interest, then focus using the appropriate microscope knob.
   6. Click Stop in the Acquisition tab to turn off the laser exposure.
3. Define the imaging parameters.
   1. Use the tick-boxes in the Channels tab to select one of the channels and click Live in the Acquisition tab to display the image of that channel on the screen. Then, using the left-click button on the mouse, adjust the slide-bars to set the range of emitted fluorescent signal that is collected, the laser power (sliding bar under Lasers heading), Gain (Master) and Digital Offset in the Channels tab to visualize the Confetti-labeled cells on the screen. Guideline values for all of these parameters are provided in Supplementary file 1B-D for RFP, YFP, and CFP. Complete this step for every channel, one by one.
      NOTE: To ensure the visualized signal is not autofluorescence, a section from a Cre negative animal can be used as a negative control during this step. As T-PMT has been combined with the RFP laser (step 7.2.1) adjusting laser power for RFP will affect T-PMT signal.
   2. Adjust the Pinhole size to increase the fluorescence detection if necessary.
      NOTE: The higher the pinhole size, the more fluorescent signal is detected in the Z-direction. A guideline for this is the resultant Airy Unit (AU), which is automatically indicated beneath the Pinhole indicator, where 1 AU is optimal for imaging quality. Increasing the AU too much will impair later analysis because it becomes more difficult to distinguish individual cells in the Z-direction).
   3. Check the setup to ensure that the recorded signals do not overlap.
      1. Click on the tick-boxes in the Channels tab to select all three channels and press the Snap button in the Acquisition tab.
      2. In the resultant image, scroll between the displayed channels by clicking the blue-highlighted boxes above each listed Channel in the Dimensions tab. Manually check for the overlap between the channels on the screen. If the signals overlap (e.g., if every red cell is also yellow), return to the beginning of step 7.3 and repeat, adjusting the parameters for the overlapping channels until they can be distinguished from each other. Ensure that for each label (i.e., RFP, YFP, or CFP), at least one clone can be visualized that contains only one of the labels.
4. Acquire image.
   1. Select the Acquisition Mode tab to specify image quality using the Frame Size, Speed, and Averaging Number values.
      NOTE: The typical settings for these experiments are indicated in Supplementary file 1A. Reducing the Zoom under the Scan Area heading within the Acquisition tab can increase the field of view without altering the time required for image acquisition.
   2. In the Acquisition tab, under the Dimensional Acquisition heading, click the Z-Stack tick-box. Open the Z-stack tab to set the interval between images in the Z-direction to 2.5 µm.
   3. To set the correct locations, select only the RFP/T-PMT in the Channels tab and click Live to visualize the red clones with a visible outline of the tissue. Set the first and last slice by clicking Set First and Set Last on the corresponding slice, adjusting the focus using the microscope knob.
   4. Click the Tile Scan tab within the Acquisition tab and enter the total number of tiles in the horizontal and vertical directions in the respective boxes.
   5. For image capture, select all the desired channels in the Channels tab, then click the Snap button to collect a single image, or the Start Experiment button to collect a Z stack/Tile scan.
5. Once the image has been acquired, click File then Save As and save the image in a file type that preserves as much information about the microscope settings as possible, to allow later review and reuse.
6. Perform further analysis using image analysis software.