Clonal Genetic Tracing using the Confetti Mouse to Study Mineralized Tissues

By genetically labeling individual cells with fluorescent proteins and following those cells over time, the Confetti Mouse has allowed researchers to reveal new insights into many biological processes. Our protocol minimizes lag time between tissue collection and imaging, can be applied to any mineralized or nonmineralized tissue and preserves fluorescence for several years. This method is very useful for all areas of regenerative medicine and developmental biology because it can be applied to different cellular populations to study their fate and their kinetics as long as a suitable Cre line is available.

Our step-by-step instructions will demonstrate the most challenging step to distinguish difference of fluorescent signals emitted by different fluorescent proteins. Begin by preparing the mouse tissue for imaging. For soft tissues and mineralized tibiae and femora from mice up to 45 days old, fix the tissue in precooled 3.7%formaldehyde PBS and incubate it at 4 degrees Celsius for six hours while rolling gently on a rotator.

For mineralized tissues such as tibiae and femora from mice over the age of 45 days, prepare one liter of 10%EDTA solution with the pH adjusted to 8.05 with sodium hydroxide and dilute the formaldehyde to 3.7%using this solution. Fix the tissue by keeping it in 3.7%formaldehyde PBS overnight at 4 degrees Celsius. On the next day, place it into the precooled formaldehyde EDTA solution and incubate it at 4 degrees Celsius while rotating for 48 hours, making sure to replace the solution three times during the incubation.

Then place the tissue into precooled 30%sucrose, making sure to fill the container to the brim, and gently rotate it overnight at four degrees Celsius. In the morning, use forceps to remove the tissue from the sucrose solution and rinse it in five milliliters of optimal cutting temperature compound, or OCT, for a few seconds. Fill a pre-labeled cryomold with OCT and position the tissue at the bottom, working quickly to avoid the sample's sitting at room temperature for too long.

Embed the sample by placing the mold on dry ice and waiting for the OCT to solidify. If the samples are not used immediately, they can be stored at minus 20 degrees Celsius. Remove the block from the mold.

Apply OCT between the top of the block and the chuck and cool it in the cryostat for at least five minutes or until the OCT completely solidifies. Precool the sample holder and the blade holder to minus 20 degrees Celsius, then place the sample in the chuck holder and the blade in the blade holder, and allow them to equilibrate for several minutes. For postnatal growth plate analysis, prepare sections that are 30 to 160 micrometers.

Use the cryostat to trim the block and cut tissue sections to a suitable thickness and collect them on slides. Then air dry the slides at room temperature until they are completely dry and store them at minus 20 degrees Celsius for up to 36 months. When ready to use the slide, remove it from the freezer and bring it to room temperature in a slide holder.

To remove the OCT, gently apply PBS onto the slide with the Pasteur pipette. For 160 micrometer thick sections, incubate the slide for 15 minutes, remove the liquid and apply fresh PBS for five more minutes. Mount the slides in a solution of 75%thiodiethanol at room temperature with 60 millimeter long cover slips.

First, select the RFP channel by clicking on it in the channels tab, which causes the details of the channel to be displayed in the light path tab. Then click the tick box beside T-PMT. Place the slide in the slide holder and position the tissue of interest directly between the light path and the objective lens.

To localize the tissue, deselect the YFP and CFP boxes under the tracks heading of the channels tab. Select the T-PMT channel in the tracks heading, then click live in the acquisition tab and increase the gain for the T-PMT channel in order to visualize the tissue selection on the screen. Adjust the position of the slide to locate the region of interest and focus using the appropriate microscope knob.

Then click stop in the acquisition tab to turn off the laser exposure. Define imaging parameters according to manuscript instructions and adjust the pinhole size if necessary. Check the setup to ensure that the recorded signals do not overlap, then select all three channels in the channels tab and press the snap button.

Scroll between the display channels in the resulting image by clicking the blue highlighted boxes above each channel in the dimensions tab and manually check for overlap between the channels on the screen. If the signals overlap, readjust the parameters until they can be distinguished from one another. This protocol was used to label chondrocytes in the epiphyseal cartilage and visualize their clonal expansion with age.

The central section was determined by visualizing the cruciate ligament and clones in the proximal tibial growth plate were imaged. Individual cells that were Col2 positive and became labeled with Confetti fluorescent proteins upon tamoxifen administration, underwent clonal expansion and subsequently remained in the growth plate. The fluorescent signal can be visualized after long-term storage.

Some sections were stored for over three years prior to preparation and imaging. Three-dimensional reconstruction was conducted automatically with image analysis software and exported as a video. One section broken during the cryosectioning step contains an area with such a high level of recombination that it would prevent clonal analysis, demonstrating one of the common problems encountered with this protocol.

Depending on your research question, using a Cre line that specifically labels your cells of interest could be very important. Therefore, we recommend checking fluorescence in those cells shortly after recombination. Using inducible Cre lines is essential because ongoing DNA recombination in noninducible Cre lines can alter the fluorescent profile of the soluted cells thereby precluding clonal analysis.

This protocol can be combined with functional perturbations such as genetically manipulated strains, pharmacological treatments, surgical interventions, as well as immuno fluorescence staining of prepared sections.