We have looked at deparaffinization and rehydration of paraffin-embedded tissue, as well as antigen-retrieval protocol and permeabilization and blocking non-specific binding on the sectioned tissue sample on the slide. In this section, we look briefly on antibody selection and antibody staining for immunohistochemistry. The selection of antibody for immunohistochemistry analysis is important for the overall success of the reaction process. This is because, if the wrong type of antibody is selected, there will be low or poor specificity for antibody binding to antigen, and this will negatively affect the outcome of the immunohistochemistry result. Therefore, to increase the possibility of high specificity and low cross-reaction of antibody with other substances, it is important that the antibody to be used is selected for an optimal result. The types of antibody to be used for the staining can either be a monoclonal antibody or a polyclonal antibody. However, the choice of which type of antibody to be used may be gotten from:
- Intensive literature search
- Checking in-house validation data of the antibody vendor
- Checking online for antibody resources and manufacturers for guide on procurement, usage, storage, and other technical assistance
When choosing the type of antibody to use for your immunohistochemistry experiment, it is important to ask yourself some important questions that will enable you to make an informed decision on which type of antibody to buy. This will also help you to know the manufacturer to buy from and which one not to patronize, because at the end of the day, all you want is the best result from your anticipated experiment. Before procuring your antibody, ask yourself the following questions:
- What is the cost of the antibody?
- What is the rate of usage of the antibody?
- How many authors have used the antibody?
- Who is the original manufacturer of the antibody?
- How available is the antibody?
- Has the antibody been validated or how was it validated?
- Has the antibody been previously used to test your question of interest?
- Do I need a polyclonal or monoclonal antibody for my experiment?
While polyclonal antibodies target multiple epitopes on the antigen, monoclonal antibody is only specific to a singular epitope. Monoclonal antibody is suitable when a homogenous population of sample is used or targeted. However, polyclonal antibody is suitable for heterogonous (different) cell population. Antibodies that are used by many researchers and in several instances are often more preferable to those used by only one author or for just a short period of time. And the cost and availability of the antibody is important because knowing this fact will help you to estimate its cost and availability anytime that you need it for your experiment. It makes no economic or scientific sense buying antibodies that are too expensive and may not be available in the future for any futuristic experiments. It is also important that researchers optimize their antibody to get a high quality staining result. The optimal condition for the primary antibody usually depends on the researchers experiment; this way, a high quality staining is obtained. When optimizing conditions for the primary antibody in immunohistochemistry, it is important to keep incubation time and temperature, and then, titrate different antibody dilutions.
PROCEDURE FOR ANTIBODY STAINING
- Add the primary antibody to the tissue sections on the slide (diluted in 1 % animal serum in PBS-T) and incubate slides at room temperature for about 1-2 hours at 4o This incubation can be continued overnight. PBS-T is phosphate buffered saline with 0.4 % Triton X-100.
- After incubation, wash the tissue sections on the slide twice with 1 % serum in PBS-T for 10 min each.
- After washing, add a biotinylated secondary antibody and incubate the sections at room temperature for 1 hour.
- Wash the tissue sections twice with 1 % serum PBS-T for 10 min each.