The Dyment Lab in the Department of Orthopaedic Surgery at the Perelman School of Medicine investigates the fundamental mechanisms that contribute to the growth and repairment of developing tissues. Over the summer, I had the opportunity to work with Professor Dyment on a project studying the time periods during tendon growth in which certain markers, such as GFP or CFP, are expressed. While the specific markers during tendon development are known, the time at which they are expressed are not. The project first came about as a result of the belief that tendon cell development needed to be better understood due to the prevalence of tendon injuries across all ages in the population. Ultimately, we hope to be able to improve tendon repair in a clinical setting.
For the project, we had to first collect samples from mice ranging across different ages specifically 4, 14, 21, and 28 days after birth. After the limbs of the mice were collected, they fixed in a solution called Formalin. Essentially, the proteins in the tissue are cross-linked to each other enabling the preservation of the tissue. After fixation, the limbs were embedded in an OCT compound, frozen, then sectioned. The sectioning of the tissue samples was done on a device called the cryostat in which we stabilize the block of tissue on a stage and cut very thin sections of about 30 micrometers. All of the collected sections were then filtered for the good cuts and glued onto a slide. These sections were first stained with DAPI, which shows the expression of GFP, CFP, and DAPI markers when imaged under our microscope. We were able to use these images to compare the expression of these different markers amongst all of our age groups (4, 14, 21, 28 days after birth). The sections underwent a second round of staining with Toluidine Blue. The images obtained from this round of staining were then merged with images from the previous round of staining to create a more informative visual. Finally, GFP and CFP intensity was recorded for each cell within the tendon/ligament midsubstance. An equivalent minimum threshold was applied and the percentage of GFP+/CFP+ cells was computed. This process was known as quantification. The final results of the quantification were then noted on an excel spreadsheet.
On a larger scale, I was able to learn the fundamental skills of research that will prove useful no matter which field I choose to pursue. Having worked with a lab partner, I was able to improve my communication skills and my ability to work efficiently in a team setting. More specifically, however, I learned how to properly use a cryostat which is a common instrument used in scientific research, the functions behind certain solutions (chitosan, DAPI, PBS), and how to properly use a Axioscan Microscope. Without a doubt, all of these technical skills will be employed in any future scientific research I hope to dive into. I am excited to see where this project ends up and I will most definitely follow the progress of it.