It is not surprising that some antibodies that give excellent results in techniques such as ELISA, WB, IP … fail when applied in immunohistochemical techniques , resulting in nonspecific (background staining) or producing very weak or null signals. This is mainly due to the fact that the reagents used in tissue fixation (eg formaldehyde) as well as the paraffin in which they are subsequently embedded, and which can produce alterations in the antigen present in the sample.
How Can We Solve This Problem?
The key is in the validation of the antibodies. The method used to test its functionality is crucial for obtaining optimal results in immunohistochemistry.
One of the strictest validation processes that guarantees 100% proper functioning of the antibodies used in immunohistochemistry is followed by the North American LifeSpan BioSciences, Inc. This process is based on a series of guidelines that we summarize below:
- Choosing the antibody: Immunohistochemistry requires the use of a secondary antibody. In order to minimize background, the primary antibody should have been produced in a species other than that of the target tissue, thus avoiding detection of endogenous immunoglobulins by the secondary. In the event that this is not possible and the primary antibody has been produced in the same species as the tissue to be studied, we will avoid this same problem if said antibody contains a tag and we can make use of a secondary anti-tag instead of an anti-Ig of the species in question.
- Determine the specificity of the antibody: In the case of antibodies for which a peptide has been used as an antigen, it is necessary to ensure that the selected amino acid sequence is unique to the target protein and that it does not repeat, for example, in proteins of the same family. We can rule out homologies by means of a BLAST. In the event that the antigen used has been a protein, we will perform an immunohistochemistry or immunocytochemistry on cell lines that overexpress the target protein, as well as cell lines that serve as a negative control.
- Fixation of the tissue: In the case of fresh and / or frozen tissues, the antigens are kept in a state similar to the native one, but when fixed with preservatives such as formalin, paraformaldehyde, alcohol, picric acid …, they are often denatured and refolded in structures that cannot be recognized by the antibody generated against the native protein. It is important to pay attention to the type of antibodies that will be used for Immunohistochemistry:
- Monoclonal antibodies for immunohistochemistry: it is very difficult for a monoclonal against the native form to recognize this protein on a fixed tissue. In some cases, the antigen conformation can be recovered using reagents like Proteinase K, although this will only work in about half of the cases. On the other hand, if we get a monoclonal that recognizes the fixed protein, we will have a practically non-existent background.
- Polyclonal Antibodies for Immunohistochemistry : The probability of success of a polyclonal far exceeds that of a monoclonal, but will consequently also produce a higher nonspecific background.
- Tissue validation: The sections are previously validated against antibodies that serve as positive controls (anti cytokeratins, vimentin, CD31, CD3, CD20 or GFAP), to ensure that antigenicity is maintained after fixation.
- Immunohistochemical validation : Each antibody is tested (in at least 4 dilutions / concentrations) on a tissue array consisting of formalin-fixed, paraffin-embedded multi-tissues. This analysis allows obtaining an overview of the behavior of the antibody in terms of sensitivity, specificity against different cell types and nonspecific background level.
Following this exhaustive validation process, the functionality of the antibody in immunohistochemistry is guaranteed. From here we propose a list of antibodies for immunohistochemistry (to see the list click here ), with more than 11,000 references that have already been validated according to these guidelines, and therefore guarantee 100% satisfaction.