Immunohistochemistry is a test in the laboratory that uses antibodies to find certain markers in a tissue sample. Fluorescent dyes and an enzyme are usually attached to the antibodies. The enzymes or dyes are activated after the antibodies attach to the markers in the tissue sample and then that marker can be seen under a microscope. Immunohistochemistry is used to help detect cancers and other diseases.
Immunohistochemistry is a type of staining that is used to find cells that are not normal, like the ones found in cancerous tumors. See here for more information about immunohistochemistry. There are specific markers at the molecular level that characterize certain cellular level events like the propagation of cells or the death of cells. IHC is also used for basic research and understanding of the dispersal and localization of different markers and proteins in different parts of the tissue. Below are the steps of using immunohistochemistry.
- 1 What is Immunohistochemistry with advantages and disadvantages?
What is Immunohistochemistry with advantages and disadvantages?
Preparing the Tissue
You can start by using tissue samples that are frozen, or they can be fixed. This is a good site to get all of the steps for preparing the tissue: https://www.ptglab.com/news/blog/a-beginners-guide-to-immunohistochemistry/. Freezing is good because it maintains the shape of the antigen markers making it easier for antibodies to bind, but it also allows small ice crystals to form in the tissue, making them harder to store long term. If you want to store your samples longer, you may want to use fixed tissue. This will allow it to be stored longer.
Retrieving the Antigen
The most common way to fix the tissue is by using formaldehyde. This causes proteins to cross-link with the tissue that causes epitopes that are recognized by the antibody to denature but also allows the shape of the tissue to stay the same. If retrieval is needed, it needs to be done before staining the sections to find the hidden epitopes. This can be done by adding heat or enzymes.
Handling the Samples
Do not let the samples dry out. Use a special tray that will help keep the tissue sections in a humid environment. You could also use a tray that has tissue soaked in water. Although there is very little risk to humans, you still need to be careful handling the samples to reduce the threat of contamination.
You will need to add a permeabilization step for most proteins. It is not necessary for all proteins like transmembrane proteins that have epitopes in the extracellular region.
A blocking solution is needed to bind to non-specific binding sites inside the tissue. This prevents non-specific binding of the antibodies to tissue parts.
Dilution of Antibody
There is usually a guide for dilutions provided with the antibody sheet. If not, you can use a dilution of one in fifty to one in three hundred as a starting point.
You need to wash off the antibody with a good wash solution. Follow a quick wash with a three-to-five-minute wash.
These are the steps that need to be followed to do immunohistochemistry. This is just a list of steps for the laymen, if you need to know more about immunohistochemistry, there are more professional websites for you to follow.
What are the Advantages of Immunohistochemistry?
The first advantage is that you can use fresh or frozen samples of tissue. IHC is also very speedily available, and it is already well established, and it has a low cost, and there is also a fast turnaround time for results. There are no live agents so there is little chance of risk to human health.
What are the Disadvantages of IHC?
The stains for IHC are not standardized throughout the world, and the cost of the equipment to do the testing is fairly expensive. Quantifying the results is not easy, and it is very susceptible to human error.
This article is just the beginning to explaining immunohistochemistry and is in no way an all-knowing guide. There are many more well-informed websites and articles about this subject that has much more knowledge. Please seek one of these more informed sites for more concrete information than this article can provide. There are also many classes that you can take to learn more about immunohistochemistry.