Research Methods: Immunohistochemistry
In research, many questions asked require us to determine if a specific protein is present in specific cells at a specific time. To answer this questions, use immunohistochemistry.Â
1) Step 1 outlines how we find the protein to begin with. IHC takes advantage of animals’ natural immune response, hence the “immuno”. If you take a protein, say human hemoglobin, and put it in a non-human, say a goat, it will recognize it as a foreign object and produce antibodies against it. You can then collect these antibodies, and boom, you have something that will bind to that protein wherever it is present. So if I’m looking for hemoglobin in human cells, I will soak a solution containing this antibody and it will bind to all the hemoglobin
2) So at this point, you have an antibody bound to a protein, but you have no way of knowing where this binding happened. This is where the secondary antibody comes in. This antibody was created in response to injection of proteins from the animal the primary antibody was taken from (in our example, goat). This ensures this secondary antibody will bind anywhere a goat protein/antibody is present. This antibody will either be florescent of biotinylated. If it’s florescent, you just shine a florescent microscope on the cells and you can see wherever the secondary antibody is bound (which will also be where the primary antibody and original protein are). However, in this example, we’re using a biotinylated antibody, which requires steps 3 and 4 to be able to see where the protein is.
3) Biotinylated means there are binding spots that ABC will bind to. ABC stands for “avidin/biotinylated enzyme complex”. This serves to amplify the signal. As you can see from the image now, there’s more than a 1:1 relationship. Several avidin/biotinylated enzyme complexes are bound to one secondary antibody. This amplification becomes beneficial when it comes to staining
4) This step is staining to determine where this complex of ABC/2°/1° antibodies are. You combine diaminobenzidine (DAB) and hydrogen peroxide (H202) which will set off a reaction that creates a brown precipitate. This “sticks” to the enzymes in the avidin/biotinylated enzyme complex, so when you wash away the excess stain, the remaining brown spots indicate the present of the protein.
The outcome of IHC can be seen in the following 2 images, but in black in white. The first image shows serotonin expression in brain cells from zebrafish that were given ketamine and fluoxetine (Prozac) co-administration. These are two antidepressants, so it is unsurprising that there is lots of staining
The second image shows IHC staining on cells from a zebrafish that had undergone two weeks of ethanol withdrawal. These fish have very little serotonin. This stain was used to conclude that fluoxetine and ketamine co-administration can prevent a deficit in serotonin synthesis in zebrafish that have undergone ethanol withdrawal.
References: Immunohistochemistry Guide from Vector Laboratories
Pittman & Hylton, 2015: DOI: 10.1016/j.pbb.2015.08.014
