Immunohistochemistry Overview

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Immunohistochemistry (IHC) is an imaging technique that allows for the visualization of proteins within a tissue section. IHC is powerful because it can provide both quantitative and qualitative information regarding the amount of protein present, its architecture, and its localization.


What is IHC?

IHC is a chromogenic imaging technique that allows one or several proteins to be visualized in a tissue section. The principle of IHC was first described in 19411, while the application based on an enzymatic reaction that leads to the deposition of a colored precipitate onto the slide was recorded almost 30 years later2. This latter protocol is what comes to mind for most scientists when thinking about IHC, as the evolution of immunofluorescent (IFM) imaging techniques has diverged over the years.


How Does It Work?

IHC is a type of immunostaining technique based on the specific recognition of a target antigen by an antibody. To begin, a 4-7 m thick tissue section is affixed to a glass slide. In order to prevent non-specific staining, the slide is blocked with a buffer containing irrelevant proteins. Next, one or more antibodies specific to the target proteins of interest are incubated on the slide.

As IHC is based on enzymatic reactions leading to the deposition of a colored precipitate onto the slide, the most important step is the choice of enzyme that is paired to the primary antibody. Either an enzyme-conjugated primary antibody or a primary-secondary pair can be used. The most common enzymes used in IHC are horseradish peroxidase (HRP) or alkaline phosphatase (AP). Different substrates produce different colored stains; the most commonly observed colors in IHC stains include brown (substrate, DAB) and red (substrate, AEC). The limit to the number of proteins that can be marked on a single slide by IHC is limited only by the number of differently colored enzyme-substrate complexes available; typically, IHC is used to visualize one or two proteins3.

After the enzyme-substrate reaction has taken place, the slide is counterstained with hematoxylin, a blue stain that marks cell nuclei. IHC slides can be viewed by light microscopes at a low magnification to view protein distribution over an entire section, or at a high magnification to analyze specific cells or tissue regions.


Why IHC?

One distinct advantage of IHC is that it produces a stain that is visible to the naked eye, without the need for equipment such as the optical filters required for IFM. Slides stained by IHC can be visualized by any light microscope. IHC can be performed manually, though automated systems that have recently been made available allow for standardization of the technique4. IHC stains can be used to determine the localization of protein within a tissue section as well as the type of cell expressing that protein; IHC is more powerful than wide field IFM for this application as the hematoxylin nuclear stain used in IHC can more clearly distinguish tissue architecture than can the nuclear stains used in IFM. IHC images can also be quantified to determine the density of cells positive for a particular protein or the percentage of marked surface area of a tissue. Due to its versatility, IHC can be adapted to projects in basic or clinical research5.

Bethyl sells a wide variety of antibodies for use in immunohistochemistry. These products have recently been used to study:

  • Previously unidentified genetic causes of a specific type of brain cancer6
  • The relationship between two unique regulatory pathways and their impact on organ development7
  • A novel nuclear protein responsible for preventing cancer8
  • A new platform designed to identify proteins associated with sarcomas9
  • The potential use of acetylation inhibitors as part of a chemotherapy regimen10



Below is the entire list of targets involved in cancer research. Can’t find what you are looking for? Bethyl offers a custom antibody service.




1. Coons A.H., Creech H.J., Jones R.N. 1941. Immunological properties of an antibody containing a fluorescent group. Experimental Biology and Medicine. 47(2): 200-202.

2. Sternberger, L. A., Hardy, P. H. Jr, Cuculis, J. J., Meyer, H. G. 1970. The unlabeled antibody enzyme method of immunohistochemistry: preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes. J. Histochem. Cytochem. 18: 315–333.

3. Nakata, T., Suzuki, N. 2012. Chromogen-based immunohistochemical method for elucidation of the coexpression of two antigens using antibodies from the same species. J Histochem Cytochem. 60(8): 611-619.

4. Prichard, J.W. 2014. Overview of automated immunohistochemistry. Arch Pathol Lab Med. 138(12): 1578-1582

5. Ramos-Vara J.A., Miller M.A. 2013. When tissue antigens and antibodies get along. Vet. Path. 51(1): 42-87.

6. Bandopadhayay P, Ramkissoon LA, Jain P, Bergthold G, Wala J, Zeid R, Schumacher SE, Urbanski L, O'Rourke R, Gibson WJ, et al. 2016. MYB-QKI rearrangements in angiocentric glioma drive tumorigenicity through a tripartite mechanism. Nat Genet. 48(3):273-82. [Bethyl antibody used: c-Myb Antibody (A304-135A)]

7. Tognon E, Kobia F, Busi I, Fumagalli, De Masi F, Vaccari T. 2016. Control of lysosomal biogenesis and Notch-dependent tissue patterning by components of the TFEB-V-ATPase axis in Drosophila melanogaster. Autophagy 12(3):499-514. [Bethyl antibody used: TFEB Antibody (A300-549A)]

8. Xue J, Chen Y, Wu Y, Wang Z, Zhou A, Zhang S, Lin K, Aldape K, Majumder S, Lu Z, et al. 2015. Tumour suppressor TRIM33 targets nuclear β-catenin degradation. Nat Commun. 6:6156. [Bethyl antibody used: TRIM33/TIF1gamma IHC Antibody (IHC-00216)]

9. Guarnerio J, Riccardi L, Taulli R, Maeda T, Wang G, Hobbs RM, Song MS, Sportoletti P, Bernardi R, Bronson RT, et al. 2015. A genetic platform to model sarcomagenesis from primary adult mesenchymal stem cells. Cancer Discov. 5(4):396-409. [Bethyl antibody used: ZBTB7/FBI-1/LRF Antibody (A300-549A

10. Wu Y, Chen H, Lu J, Zhang M, Zhang R, Duan T, Wang X, Huang J, Kang T. 2015. Acetylation-dependent function of human single-stranded DNA binding protein Nucleic Acids Res. 43(16):7878-87. [Bethyl antibodies used: p300 IHC Antibody (IHC-00028) and SSB1 IHC Antibody (IHC-00403)]