I love imaging. Especially imaging using antibodies.
I spent a large amount of time recently creating images of differentiated human iPS cells... and I loved it.
A couple of weeks ago, I was giving a plate of undifferentiated stem cells to practice passaging and removing unwanted differentiation. I noticed that there was a very cool-looking neural field developing on the plate, and decided that instead of removing the developing neurons, I would allow them to continue to grow for a couple of days and then do some imaging of them. I have grown to love the microscope over the course of this semester (last semester I wasn't so keen to use it... I found it frustrating to find what I was looking for. It's amazing what a little practice and a much more advanced microscope can do.) I also decided that if I was going to do some imaging, perhaps I would stain them with DAPI in order to visualize their nuclei. I had only done so with CHO cells in the past, and really loved the results. I knew stem cells would probably much more difficult to deal with, not to mention that the cells I wished to stain and image were still developing, so they would be extremely delicate.
As I went through the staining process, I noticed that it looked like some of the cells may have been flushed off the plate, despite what I thought was my very careful technique. I suppose I underestimated how careful I needed to be because when I checked the cells under the microscope when the staining process was finished, I could not find my neural fields. I felt quite defeated. I spent the weekend thinking about those cells and the images I wanted to get from them, but wasn't able to. I have to say, it made me sad. I had this amazing opportunity and I felt as though I blew it. In the post-bacc intensive program, we don't usually get to play around much with stem cells.
When I came in on Monday, I was excited to find that I would have yet another opportunity to stain some stem cells! In fact, not only was I going to be able to use DAPI for the nuclei, but I would be using another technique to stain beta-tubulin in the neurons using Alexafluor 488. The Alexafluor is conjugated to a secondary antibody which binds to a primary antibody which targets the beta-tubulin in the axons of the neural cells. When imaged under a specific wavelength, the Alexafluor glows green. Because of my prior failure, I had learned my lesson. I took my time and paid very close attention to my technique, pipetting the reagents as gently and carefully as possible. The following are some examples of the images I created using this staining technique:
I was so excited about the way these images turned out that I immediately wanted to do more!
I'm currently working on my independent project for the intensive program and I've decided to use ICC to characterize the three different derm layers of differentiated human embryonic stem cells as well as pluripotency of undifferentiated induced pluripotent stem cells. I spent the last week culturing my cells, figuring out the best cells and matrix to use in order to get the cells to plate to glass coverslips. They don't like glass, but I finally got them to stick. Yesterday I did a practice run, treating some of my cells with a stain that targets mitochondria and makes them glow red. The following are some images I took of those cells:
After I treat my cells using this mito stain, I will use antibodies to counter-stain them green, using a different antibody to target specific biomarkers for endoderm, mesoderm, ectoderm and pluripotency. Then, I'll do a blue DAPI stain to visualize the nuclei and give some context to the cells. Wish me luck!