To begin, fix the limbs of an anesthetized mouse with adhesive tape. Hold the skin of the abdomen upward and carefully cut along the circumference of the thorax to fully expose it. Make a small cut in the right atrium with ophthalmic scissors and immediately insert a perfusion 22 gauge needle at the apex of the left ventricle.
Then push 30 to 50 milliliters of cardiac perfusion fluid through the syringe. After the outflow fluid from the right atrium becomes completely clear, infuse an equal volume of 4%PFA solution. Dissect the tissues and organs and fix them overnight with 4%PFA at four degrees Celsius.
To induce tissue decalcification. place the fixed heart tissue in 10 milliliters of EDTA solution on a shaking table at 37 degrees Celsius. Incubate for about two days and change the fluid daily.
Place the fixed calvaria bones in 20 milliliters of 25%Quadrol solution for tissue decolorization. Incubate on a shaking table at 37 degrees Celsius for one day and change the fluid once. Next, place the tissue sequentially in 30%Tertiary-Butanol or TB solution.
Then 50%TB solution, and then 70%TB solution to perform gradient degreasing. Subsequently dehydrate the samples and TB PEG solution for six hours on a shaking table at 37 degrees Celsius. Finally, place the completely dehydrated tissue in BB PEG solution on a shaking table at 37 degrees Celsius.
After two to four hours of incubation, the tissue will turn transparent. The PEGASOS Tissue Clearing Method resulted in transparent tissue. Cleared tissue with whole mount EDU staining showed that the labeling was efficient and well preserved.
In the control group, several EDU positive cells were diffusely distributed throughout the sutures. After one day of suture expansion, proliferating cells were observed in the middle and edges of the suture. As the suture widened, the number of proliferating cells decreased over time.
The green labeled cells were small round cells in the bone marrow, which differed from the EDU positive suture cells.
