The last two methods allowed me to assess the functionality and calcification of the aortic valves. In my next procedure, I measured pro-osteogenesis tendencies by conducting an immunohistological analysis. Calcification is a part of bone growth (osteogenesis), so another way to prove the existence of calcification would be to measure the pro-osteogenic tendencies of the valve. I identified a pro-osteogenic marker gene called osterix, which I would measure.
I took my frozen mice aortic valve cross-sections and stained them with a fluorescent dye that had antibodies that attached to certain parts of the tissue and make it fluoresce when prompted. Under a confocal, a special microscope that uses lasers, I was able to see the different parts of the tissue. It looked like this:
The first picture (blue dots) show the nuclei of the aortic valve, the second picture (red dots) highlights the osterix, the third picture (green dots) shows the endothelial cells, and the last picture shows them all combined. To analyze these pictures, I calculated the osterix levels of the aortic valve samples and compared them against each other to see if SIRT6 reductions had any impact on pro-osteogenesis.