A new month has arrived, which means a new ‘Cellfie’ of the month!
So this is just my second post in this series where I want to give you a bit more of a behind the scenes look into PhD life by showing you some of the sights I see down a microscope and talk all things involved in keeping my cells happy and healthy ☺️. In my previous ‘Cellfie’ of the month blog, I introduced you to my favourite image that I had taken so far of my embryonic stem cells using the technique called immunocytochemistry, and introduced you to the pattern we see if the protein we are looking at is expressed on the surface of the cells.
In this blog, I am going to show you another one of my images, but this time I am looking at the expression of the protein OCT4, which as promised shows a different expression pattern!
OCT4 is a really important protein for my PhD research as it is one of the three core pluripotency factors – so when a cell is expressing OCT4, it is classed as pluripotent and could become any cell type in your body! So, for my research, as I am looking at ways to keep embryonic stem cells as stem cells (or pluripotent!), I look at the expression levels of OCT4 after most of the experiments I do to see how it changes. If the OCT4 expression goes down, we know that the conditions we used in that experiment are not good for stem cells. If OCT4 expression increases, those conditions are good to try and keep stem cells pluripotent!
OCT4 is, also, known as a transcription factor. This means it can bind to certain regions of DNA and ‘turn on’ a particular gene. DNA is stored in the nucleus of your cells, so OCT4 is also going to be found in the nucleus of my embryonic stem cells.
If you can remember from last time, the blue staining is the DAPI stain. This is added to our cells to make their nuclei glow blue under the microscope. Using this immunocytochemistry technique, I have also added an antibody to these cells that will stick to any OCT4 that is in the cells, which is shown here in green. Notice anything about these two images?
In the science research world, our ultimate aim is to get our research published. In these publications, we might need to add some of these images, but they need to be presented in certain ways. So, in my last ‘Cellfie’ blog post, I introduced the scale bar – a small line in the bottom of your image that is a reference point so the reader knows how big the cells they are looking at are. Last time I also showed you to the merged image, or the overlay. The aim of the merged image is to confirm where a protein is expressed. For example, in the last post where I showed you the overlay for a surface marker – there were the blue dots that were the nuclei of my cells, and green areas around them. Here, we have blue dots which are again the nuclei of my cells, but we also have green dots when we are looking for OCT4 in the images above. When we merge the images together we get this:
The blue DAPI staining and the green OCT4 staining overlap – which just confirms that OCT4 is in the nucleus of my cells as they completely overlap. Hopefully you can see how this is different to the staining in my last ‘Cellfie’ post.
So – my stem cells are expressing lots of OCT4 in the nucleus. So by showing these images in my thesis or any publications I have demonstrated that the cells I am using are pluripotent!
Another ‘cellfie’ post done! I hope you enjoyed. I would love to hear your feedback! I have something a little different up my sleeve for the next one so watch this space.