The world of the Science PhD student is shrouded in mystery. What sorts of things do we actually do? How do we spend our days? What experiments do we do?
I shall offer you a bit of insight by showing you my typical day in the lab… at the moment! Currently I am doing more in my cell biology lab, so that will be the focus of this post, but when I do anything exciting with my Tomatoes I will do a post to show you some of those experiments too.
My day in the lab starts at about 9.30am. This is pretty flexible, sometimes I am in for 8.30am, sometimes I may come in after 10am- it all depends on what I have to do in the lab, and how easy it was to get out of bed in the morning!
First things first: preparation of any media I need. Its always good before you start a big experiment to actually check you have all the things you need first.
Today I am doing a immunohistochemistry stain. Staining is a common method used in biology- we use it to visualise specific cellular components, such as proteins, within tissues.
Immunohistochemistry is a particular type of staining that uses antibodies to bind to a specific antigen (e.g a protein) . Antibodies are highly specific, and so will only bind to the antigen of interest within a tissue. The interaction between the antibody and antigen is visualised using a chromagen. For my staining, I am using a substrate called DAB (3,3′-Diaminobenzidine). In DAB staining the antibody is linked to a peroxidase enzyme, when hydrogen peroxide is added with DAB, the enzyme breaks the DAB to a brown precipitate– As only the ‘target’ will have the antibody attached, this brown colour is specifically located to that area.
Starting the experiment
First thing to do is outline all my prepared slide sections with a hydrophobic marker, and wash the slides in some PBS.
Label all the slides and add block. Tissues can have endogenous peroxidase activity- as the DAB stain depends on the action on a peroxidase attached to my antibody- I need to quench endogenous peroxidase activity. This will help reduce any background staining.
Wash all the slides in PBS again
Add the first antibody- this is the antibody which binds to the antigen. The slides are left to incubate with the primary antibody in this self made incubation chamber for 90 minutes.
Whilst my slides are incubating, its a chance for me to grab some coffee, and catch up on some extra work- today I am going through my micro-array data and trying to make sense of it all.
Time to remove the primary antibody, wash the slides, and add the secondary antibody. The secondary antibody will bind specifically to the primary antibody, and has Biotin ( a small vitamin) attached to it. More on this later.
The slides with the secondary antibody are put back in the incubation and chamber and incubated for another 1hr 30, giving me some time to have some lunch…
30 minutes before the end of the secondary antibody incubation I have to make up a ‘ABC-HRP’ solution. The biotin on the secondary antibody has a very strong affinity for Avidin, a large glycoprotein. In this solution Avidin is mixed with biotinylated-HRP, forming a large structure of avidin-biotin-enzyme. After the rest of the secondary antibody- incubation, and washing of slides, this solution is then added and incubated for 20 minutes.
The Avidin-enzyme complex binds to all the biotin on the secondary antibody- this is basically a way to get a greater concentration of enzyme at the antigen site and so will increase the overall signal I get. (Click here for more info on how this works)
The slides are washed again, and then the DAB stain solution is added. This solution reacts with the peroxidase, forming the colour. I added Nickel to the DAB solution, so that instead of a brown colour being produced, a black colour is produced.
After 10 minutes of incubation, the slides are put in water, to stop further development of DAB and a counter stain is added. The counterstain colours the rest of the tissue, this makes it much easier to visualise the tissue, and see exactly where the DAB staining is.
Finally, after counterstaining the slides, and dehydrating and clearing the slides (to help the overall appearance of the sections) it is time to mount the slides with coverslips.
Now all the slides are mounted, I can have a look at all my hardwork on the microscope, take some photos, and see if the staining worked!
Back to the write up office to sort out all the photos, pop them in a presentation and analyse them before sending them to my supervisor.
My final task before going home is to check through any emails I have got through the day, plan my next days experiments, and have another play around with my microarray data.
And that was what I did with my day yesterday! I hope you guys enjoyed looking at a day in my lab-life, and hopefully my explanations of what I was doing hasn’t left you too confused! I will leave some more links to explain IHC more below.