After freezing dozens of mouse aortic valve slices, I conducted our first method of investigating SIRT6’s effects on CAVD: Alizarin Red stains. The purpose of this procedure is to detect calcification by highlighting calcium deposits in the frozen aortic valves. Since we know that SIRT6 reductions happen as age increases and that increased age leads to calcification, we can safely assume that SIRT6 reductions would lead to increased calcification. Based on this knowledge, I hypothesized increases in calcification in mice with SIRT6 mutations and decreases in calcification in wild type mice.
To briefly summarize, the process of Alizarin Red stain is completed by soaking the frozen cross-sections in an Alizarin Red solution first. I may or may not have forgotten to wear gloves while handling red staining solution and been stuck with stained hands for a while. The soaked samples look like this:
Then, I soaked the samples in a series of other solutions (normal buffer formalin, 1x PBS, acetone, acetone-xylene, and xylene) that help Alizarin Red react with the calcium. The Alizarin Red stain will bind to calcium in the aortic valves and under the microscope, it will show up as orange-red dots, which are actually kind of pretty:
In order to analyze the calcification in each valve, I used a microscope with a camera and took pictures of each sample aortic valve. While taking photos, I made sure to keep the lighting, focus, and zoom conditions uniform to eliminate any confounding variables that would affect our analysis. I took three photos of each valve, so I could average our analysis and find a number with a minimized error during analysis. Using Photoshop, I separated the leaflets from the whole valve, so I could measure how much calcium was on the leaflet itself. I was able to count the exact number of dark red pixels (pixels that showed calcification) in the leaflet photo. I found the ratio between the number of calcium pixels and the total number of valve pixels to measure the amount of calcium burden on the sample valve. My Photoshop analysis looked something like this:
I spent three days just completing the Photoshop part of the analysis. After I had all my numbers, I averaged the data for the sets of three pictures to get my final calcium burden for the valve as a whole. I separated the samples by the type of mouse (Young WT, Old WT, Young Cre+, and Old Cre+) and averaged all the data in each set, so I could compare the calcium burden in the different groups.
Doing the Alizarin Red stain was lengthy and tedious, but the end result was so satisfying.