An Introduction to Immunohistochemistry

Immunohistochemistry (IHC) is an assay that utilizes the biological collaboration of Histology and Immunology. The biochemical interactions between an antibody and antigen permit visual distribution and localization of antibody–antigen interactions. This allows morphology, antigen intensity, and spatial relationships to be determined.

We offer high-quality IHC assays onsite to give you a time-efficient read-out on quantitative and qualitative data. With our in-house IHC services, data can be produced expeditiously that reveals the following:

• Efficacy of your compound in target tissues
• Location of biomarkers in tissues
• Incidence of tumor metastasis
• Occurrence of tissue transformation into tumors
• Origin of tumor tissue
• Prognosis of the animals in the study

Steps to Performing Immunohistochemistry

Fig. 1: IHC Fixation and Epitope Retrieval*

IHC results are obtained through a series of sample preparations that allow manipulation of the tissue for antigen discovery. The tissue is surgically removed and placed into a fixative such as NBF or PFA. During fixation, the tissue undergoes a chemical reaction where nucleic acids and amines form semi-reversible cross links composed of methylene bridges (fig. 1). The cross links create cell and tissue stability and protect the tissue from decomposition and damage from proteolytic enzymes. Fixed tissues can then be stored in paraffin allowing them to be used at any future time for IHC.

Samples that have been fixed must undergo a method of antigen retrieval in order to detect the antigens. After sectioning and mounting on slides, the tissues must be rehydrated to permit antibody-epitope interactions. The most common epitope retrieval method is heat-induced epitope retrieval (HIER), which utilizes a combination of temperature, pH, and time to gently remove cross bridges and expose the antigens of interest.

Once the antigen has been exposed for investigation, the following general steps (fig. 2) are followed:

Fig. 2: IHC General Steps

• Blocking – Reactive sites on proteins that are not the protein of interest and endogenous peroxidases can create a false positive signal. To avoid this, multiple blocking steps are utilized to avoid non-specific binding.
• Detection – Antibody-mediated detection is achieved through either fluorescence or chromogenic means. Direct fluorescence detection (fig. 3) involves the use of a primary antibody conjugated to a fluorophore. To detect antigens that are not abundantly expressed, indirect fluorescence detection (fig. 3) amplifies the signal by using a fluorophore-conjugated secondary antibody. Chromogen detection is made possible with enzyme-conjugated secondary antibodies and a substrate that forms a colored precipitate on the tissue.

Fig. 3: IHC Indirect Detection and Direct Detection Diagram**

• Counterstain – These chemical stains are used to help identify cellular features (such as the nucleus with Hematoxylin and DAPI) as well as create a contrast between the positive antibody staining and the general organelle staining.
• Imaging – Microscopy is done for data comparison, qualitative analysis, and basic visualization of the antigen. We utilize the Aperio VERSA scanner to allow for greater detail and fine detection of antigens that are expressed at very low levels (fig. 4).

Figure 4a-f: a) KI67 positive nuclei on tonsil, b) AE1/AE3+5D3 cytoplasmic staining on Mouse Skin, c) EGFR positive staining on placenta, d) HER2 positive cytoplasm on BT474 tumor, e) PR positive nuclei on BT474 tumor, f) AE1/AE3+5D3 on epithelium of mouse tongue
 

IHC has proven to be an invaluable assay in the areas of research and drug discovery. We stay up to date on the most valuable techniques, detection, and analysis to best meet your needs. If you would like to learn more about how we can help support your study through Immunohistochemistry, please contact us so that we can discuss the best options for you.

Additional Resources:
View our IHC Brochure
View our Validated IHC Markers

*Illustration credit: https://www.nationaldiagnostics.com/histology/article/aldehyde-fixatives

**Illustration credit: http://www.leicabiosystems.com/pathologyleaders/an-animated-guide-to-immunohistochemistry/