Conducting an IHC Study - Factors to Consider: Part 1

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.

Factors to consider before conducting an IHC study:

Target of Interest
Determine the target of interest, and tissue species and tissue type to be stained.

Availability of Primary Antibodies
Determine if there are any commercially available primary antibodies against that specific target through literature searches or antibody supplier searches, otherwise it may need to be produced in-house. See the article “Conducting an IHC Study – Factors to Consider: Part 2 for the criteria in choosing an appropriate primary antibody. If none are available, an alternate target of interest may be chosen depending on the question being addressed. Identify the best antibody from the panel of antibodies sourced.

Type of Sample Processing
Determine whether the samples should undergo paraffin or frozen processing by identifying if the antibody is suitable for paraffin or frozen samples or both. Some antibodies for specific antigens are known to only work on frozen due to the retention of the immunoreactivity of the antigen. In other words, the antibody/antigen may dictate whether paraffin or frozen processing is required. If an antibody works for both types of samples, paraffin is preferred over frozen due to better tissue morphology and more optimal long-term storage, since paraffin blocks can be stored at room temperature, whereas frozen blocks need special storage at -20°C or -80°C (also, frozen blocks can form ice crystals with time). While paraffin has its advantages, one drawback is that antigens may be destroyed during paraffin processing, and antigen retrieval may be required to reverse the cross-links between the fixative and antigen in the tissue. 

In the case where one has already chosen the type of processing before requesting IHC staining, the antibody selected must be suitable for that type of processing.

Type of Detection System
Determine whether to use chromogenic or immunofluorescence detection system. 

Protocol Development
A.)   Once one has considered whether or not to conduct frozen or paraffin processing, determined the suitability of the primary antibody, and chosen either chromogenic or  immunofluorescence detection, one of four general protocols should be followed:

  • Frozen sections and chromogenic detection
  • Frozen sections and immunofluorescence detection
  • Paraffin sections and chromogenic detection
  • Paraffin sections and immunofluorescence detection

Optimize the protocol on the positive and negative tissue controls first, and then the experimental tissues.

B.)  There are a few factors to consider when conducting antibody optimization. For frozen, one needs to consider the post-fixation methods (Acetone, Methanol, Acetone/Methanol, or Formalin) and the permeablization method (Triton) if the antigen is intracellular. For paraffin, one needs to decide on the antigen retrieval methods (heat-induced epitope retrieval, HIER, vs. proteolytic-induced epitope retrieval, PIER).

For primary antibody dilution, a 3-4 series dilution must occur (1 dilution below, 1 dilution within, and 1 dilution above the recommended dilution range provided by the antibody vendor) with either 1 hour or overnight incubation. For secondary antibodies, it must be ensured that the secondary antibody is raised against the host species of the primary antibody.  Depending on the detection system used, one can use biotin, enzyme, or fluorescence conjugated secondary antibodies.

Antibody Interpretation
Study the optimization slides and determine if there is positive staining. A positive signal exists when the colorimetric/fluorescent staining is at the expected region of the tissue and is localized in the expected cellular compartment. Therefore, it is important to do background research on these two aspects to aid in the interpretation of the staining.

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.

Conducting an IHC Study - Factors to Consider: Part II