It wouldn’t be a complete laboratory experience if everything went perfectly all of the time. Some challenges that I encountered were not evident until the data came in, other challenges occurred and were evident in the middle of an experiment and required attention before we could proceed.
It was my first time doing a particular antibody stain on rat lung tissue samples embedded in paraffin on a glass slide. I had done this particular stain on human lung tissue embedded in paraffin a few times before after figuring out what concentration of the antibody was needed. I proceeded to use the antibody concentration that worked best for the human lung tissue samples, along with a few other more diluted concentrations. The goal was to figure out what concentration was needed to avoid non-specific binding of the antibody. Long story short, the concentrations that worked in human lung tissue with overnight incubation were too low for the rat lung tissue with one-hour incubation; there was little to no stain in the rat lung tissue. I then completed another stain on the rat lung tissue with a higher antibody concentration with two-hour incubation; this stain was successful and produced specific binding of the antibody.
The biggest challenge did not end up being the pulmonary artery isolation that I’ve mentioned in other blog posts, but the pressure myograph system itself. The first week or so the myograph worked well, but then we started encountering a reverse-flow issue. The myograph is supposed to pull the solution from the P1 bottle, pass the solution through the system and artery, and proceed to dump the waste into the P2 bottle. Instead, the myograph was pulling air from the P2 bottle, pushing the air through the system and artery, then into the P1 bottle. The big issue with this? If air bubbles are introduced into the artery, they can damage the innermost cell layer, the endothelial cell layer, and cause the artery to not respond to stimuli in a physiological way or respond at all. To combat this issue, I had to manually push the solution through the P2 line when the bubble was getting near the chamber. This is not a permanent fix, but it was all we could do without damaging the vessel by increasing the pressure high enough for the system to correct the flow direction. The system in reverse flow not only runs the risk of a bubble being introduced it also has a decreased capability of stabilizing the pressure. When in reverse flow, there was a pressure difference between the P1 and P2 side, this lead to inconsistent pressure being placed on the artery. Most of our myograph experiments ended up being plagued with the reverse flow of the solution, so after careful observation to ensure that it was not a user error, we requested that the myograph be sent for maintenance to hopefully fix the reverse flow problem. The myograph will be sent off after I leave the lab this summer so I will be unable to report whether or not the problem is solved.
A small challenge that accompanied the pulmonary artery isolation is that there is only one segment of the artery in the left lung that does not contain branches. Branches that are not addressed in a myograph mount lead to pressure leaks. To use segments that are deeper into the lung, I had to tie off the side branches so that there were not pressurization issues. I found it best to have the artery over a white background so that visualization of the black 11-0 suture loops is clear. Trimming the ends of the tightened loop also allowed the myograph video camera to capture the outer diameter measurement of the artery better.
Pictured above is a segment of the left pulmonary artery (rat) with the side branches tied off.
Other than the antibody stain and myograph experiments, I did not encounter many other challenges. I gained more experience with handling rats; they like to grab onto everything when you try to pick them up. I always try to stay alert and observe everything closely so that if a problem does arise, I am able to react before a larger problem forms. A lot of the experiments and techniques that I performed would not successful is any error occurred. Despite the stress that accompanied some procedures, I enjoyed doing them, they were the most gratifying to get results from.