Previously, we looked at deparaffinization and rehydration of paraffin-embedded tissue, as well as antigen-retrieval protocol. Both deparaffinization and rehydration of paraffin-embedded tissue and antigen-retrieval are important process in the immunohistochemistry staining of paraffin-embedded tissue. In this section, we look at permeabilization and blocking non-specific binding on the sectioned tissue sample on the slide. Permeabilization and blocking non-specific ionic binding is another important process involved in the immunohistochemistry staining of paraffin-embedded tissue. Blocking is an important part of any immunohistochemical staining technique. It helps to prevent non-specific ionic binding of antibodies to the tissue of the slide. More so, blocking helps to prevent the non-specific binding of other chemical reagents to the test tissue section on the slide. Van der Waals interactions and the interaction of some specific amino acid groups are some of the non-specific ionic binding that may occur. This reaction should be prevented from happening using a blocking agent. Why is blocking important in the immunohistochemistry technique? Blocking is important because even if the test antibody for the immunohistochemistry technique has a high-specificity for the target site, some intermolecular forces that may arise during the reaction process may promote non-specific ionic binding to other molecules present during the reaction steps. More so, the occurrence of non-specific binding during the reaction process may also prevent the proper visualization of the antigen-antibody complex formed. If non-specific ionic binding occurs, it will prevent the researcher from seeing the binding of the antigen to the antibody during microscopy.
Therefore, a blocking agent should be used to prevent the non-specific ionic binding. A blocking step should always be incorporated in the immunohistochemistry technique prior to incubation of the tissue sample on the slide with the primary antibody of interest in order to diminish or prevent non-specific bonding. Examples of blocking agents used to achieve blocking during immunohistochemistry straining technique include:
- Bovine serum albumin (BSA)
The type of blocking agent to be used for blocking analysis is largely dependent on the type of tissue to be processed for immunohistochemistry analysis. When using alkaline phosphatase or horseradish peroxidase (HRP)–conjugated antibody for detection, the endogenous level of the enzyme used in the conjugated antibody have to be blocked. This applies for tissues such as intestine, kidney, lymphoid tissues (such as lymph nodes) and liver. Another type of molecule that can affect the specificity and integrity of the antibody-antigen reaction is endogenous biotin. However, endogenous biotin is high in certain types of samples / tissues such as kidney, brain and liver. And in such samples that are very rich in endogenous biotin, blocking non-specific ionic binding is crucial to the success of the experiment, especially if you are working with certain types of detection kits such as the avidin-biotin detection system.
Important steps to note in permeabilization and blocking non-specific binding
- To block endogenous peroxidase activity, first quench the tissue sections with about 2.5-3.0 % hydrogen peroxide in methanol. This should last for about 15-20 min.
- Wash the tissue sections in distilled water for about 5 min. Do this step twice.
- Wash the tissue sections twice with 1 % animal serum in phosphate buffered saline (PBS) with 0.4 % Triton X-100 (PBS-T). This step helps to permeabilize the cells or tissue sections on the slide. AND THE TYPE OF ANIMAL SERUM TO USE IS LARGELY DEPENDENT ON THE HOST OF THE SECONDARY ANTIBODY. FOR EXAMPLE, WHEN USING A GOAT ANTI-MOUSE SECONDARY ANTIBODY, MAKE SURE TO USE A GOAT SERUM FOR THE BLOCKING ANALYSIS.
- Incubate the sectioned tissue with 5 % animal serum in PBS-T for about 30 min at room temperature. This step helps to block any other non-specific ionic binding.