Conducting an IHC Study - Factors to Consider: Part II

Immunohistochemistry (IHC) is a method for demonstrating the presence and location of proteins in tissue. IHC enables the observation of processes in the context of intact tissue, which is especially useful for assessing the progression and treatment of certain diseases. Immunohistochemical staining is accomplished with antibodies that recognize the target protein. Since antibodies are highly specific, the antibody will only bind to the protein of interest. The antibody and antigen interaction is then visualized using chromogenic detection.

Finding an Appropriate Primary Antibody

As discussed in the article “Conducting an IHC Study - Factors to Consider: Part 1”, when conducting immunohistochemistry, one must first consider the target of interest, availability of primary antibodies, type of sample processing and detection system, protocol development, and antibody interpretation; Once these steps are complete, identify the appropriate primary antibody while considering the following factors:

1. Specificity: Does the antibody react with the target of interest? Is the antibody produced against the intracellular or extracellular domains of the protein? Is the antibody generated against an immunogen that is a full-length or truncated protein?

2. Application: Is the antibody suitable for IHC paraffin or IHC frozen embedded samples for chromogenic or immunofluorescence detection?  Some antibodies are suitable for one or the other, or both. Some antibodies for specific antigens are known to only work on frozen. In other words, the antibody/antigen may dictate whether paraffin or frozen processing is required.

3. Species Reactivity: The antibody must be able to react with the tissue species of interest. Some antibodies may react with multiple species due to similar amino acid sequence homology. 

4. Host Species: Avoid choosing the same host species as the tissue species since this will lead to endogenous immunoglobulin background. For example, the anti-mouse secondary antibody will bind to endogenous mouse immunoglobulin in the mouse tissue causing false-positives. In this case, it would be optimal to find an antibody produced in other species besides mouse.

5. Cellular Localization: Is the target of interest expressed in the nucleus, cytoplasm, or the cell membrane? This information will indicate whether there is true positive staining. For example, if the observed and expected cellular localization of the target differs, the staining is false.

6. Product Citation/References: Aim to find antibodies used in references by reviewing the antibody specification sheet and consulting online sources such as Pubmed, Labome, Google Scholar, etc. This will provide a rough framework of the optimized conditions such as antigen retrieval method and primary antibody dilution used by others, and what the staining should look like under optimized conditions. Ideally the references will have conducted antibody staining on the same tissue type and species, and with the detection method that will be used.

7. Positive/Negative Tissue Controls:  It is important to find the appropriate tissue controls for the target of interest. Ideally these tissue controls should be prepared in the same way as the experimental samples. Staining in the appropriate regions of the positive control tissue indicates that the IHC protocol is working properly. No staining in the negative control tissue indicates that the antibody is specific.

8. Antibody Vendor: Is the antibody vendor reputable? It is ideal to choose vendors that manufacture their own antibodies, have validated these antibodies in-house, and have provided sufficient and relevant information on the antibody specification sheets such as optimal staining conditions and product citations. Avoid vendors that purchase and re-sell antibodies if they do not provide sufficient information on their antibodies.

Wax-it Histology Services offers a range of IHC services to its clients, such as conducting background literature research and sourcing of optimal antibodies, antibody optimization, cross-reactivity studies, protocol development, routine IHC staining (manual and automated) and slide analysis (digital microscopy imaging, digital whole slide scanning, staining quantification, pathological scoring/analysis, and report generation). For more information, contact us via email at or by phone at 604.822.1595.

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