Fixation on Histology

IHC Past, Present, and Future

IHC StainToday, immunohistochemistry (IHC) is an essential and routinely used ancillary tool in anatomic pathology for clinical diagnostics and research laboratories. Over the years there have been great advancements in IHC with cancer research and clinical diagnosis, which has improved accuracy, precision, prognosis, and treatment for patients and we can expect that even greater advancements are on the horizon. As we anticipate the future of IHC, let’s take a look back at it’s journey.  

IHC has existed since 1931, but it wasn’t until 1941 that the first IHC study was reported by Coon and his colleagues. Coon experimented with the idea of using the antibody to detect the antigen in a cell. He and his colleagues identified pneumococcal antigens in a tissue section using a fluorescent labeled antibody. The fluorochrome label used was called
Fluorescein Isothiocyanate (FITC)(2). This technique was used for decades, but there were some drawbacks. One of which was the fluorescein faded as sections were viewed under the fluorescence microscope. Since then, permanent fluorescent compounds have been made. 

As researchers continued to find the best detection system that was highly sensitive, without non-specific staining, the era of IHC automation began to develop. Researchers began experimenting with detection techniques like direct immunolabeling, which is a one-step protocol as seen in Coon’s study. Since this labeling technique was not as sensitive and it produced non-specific background staining, the indirect detection method was developed. Thirty years later, further indirect detection method was invented, Peroxidase anti-peroxidase complex. This technique was pioneered by Sternberger in 1970. It presented increased sensitivity and decreased background staining. Following this technique in the 1980s Dr. Hsu developed the 3 -step protocol Avidin-Biotin Complex (ABC) and later Labeled Streptavidin Biotin (LSAB) was created. These methods are still used today, but their drawback is the potential for false positive staining due to the presence of endogenous biotin. Over time, and through much research and collaborations by scientists, a more improved detection technique was developed, the polymer-based detection method. It increased sensitivity, specificity, and avoided endogenous biotin.  

The Polymer based technique can be labeled with either enzyme, Horseradish Peroxidase (HRP) and Alkaline Phosphatase (AP), a fluorochrome, such as FITC or a gold label. Currently, HRP and AP Polymer-based detection is widely used in IHC because of its high specificity, sensitivity, and faster staining method. The chromogens of choice are DAB and AEC. HRP commonly uses the DAB chromogen, which when in the presence of a peroxidase enzyme precipitates into a brown product or AEC chromogen which precipitates a red color. AP commonly use a Fast Red chromogen, when reacted with alkaline phosphatase it produces an insoluble red precipitate. AP is great for double staining. 

As IHC progressed through the years we are no longer limited in using one chromogen to detect one antigen in a tissue section. We can now stain multiple antigens at the same time in a section using different chromogens, this technique is referred to as multiplexing IHC or multiplexing IF when using fluorescent compounds. 

Multiplexing is very significant in retrieving information in a limited sample. It concurrently identifies specific proteins or molecular abnormalities, to determine the spatial distribution and activation state of immune cells. (4) Chromogenic IHC can multiplex up to three protein markers in a tissue sample, whereas fluorescent detections can detect more markers. Multiplex IHC/IF has its limitation with overlap of multiple dyes or color mixing which makes it difficult to interpret results. However, through the latest innovations of combining IHC and mass spectroscopy it has enabled high order multiplexing and analysis. Due to the discovery of multiplexing IHC, it has ushered in the era of digital pathology and image analysis. 

Whole slide image scanning is approved by the FDA for primary diagnosis and we can now scan slides to share at tumor board meetings and use the photos as a teaching tool.  With digitization it allows remote analysis and addresses the issues of subjective bias during manual cell count. The development of image analysis software tools identifies cells and tissue compartments giving cell counts and staining intensity (3). All of which is helping IHC meet the needs of our current data driven medicine culture. 

Looking ahead, Mass Spectrometry-IHC is the future of IHC.  There are two research groups that developed two different techniques with similar approaches, scanning mass cytometry (SMC) and multiplexed ion-beam imaging (MIBI). Both methods use antibodies labeled with a pure metal tag, which presents decipherable peaks from the mass measurements of the metals. This allows for the presence, location, and abundance of target proteins to be read. 

We cannot talk about MS-IHC without mentioning CyToF. SMC is an extension of CyToF in which laser ablation of tissue is coupled with plasma time of flight mass spectrometry. It allows for the quantification of intracellular and extracellular components. Once the cell is vaporized with a laser to release the electrons from the metal to form an ion, it is read by a mass cytometer by separating the ions based on its mass. The deflector counts the ions quantifying the target proteins (5). 

The MIBI technology is a high-definition spatial proteomics platform that generates a highly multiplexed image and spatially resolute single-cell profile of the tissue microenvironment. This technology uses a scanning ion beam to sample and liberate the ions detected using a magnetic sensor mass spectrometer. It requires a specialize setup with a vacuum and a multiple detector MS. A 2-D image of the detected heavy metal ions are created by an imaging software. This technology allows a high-definition analysis of the tissue microenvironment. Giving an insight to the disease state and patient’s response to treatment. The downside is that you can only detect seven targets per scan (5) . 

Technological advancements over the years have revolutionized IHC. With advancements in automation, multiplexing and image analysis it has transformed the outlook of IHC with providing high quality care and drug development. I look forward to seeing the new developments and what the future holds for IHC in MS-IHC. 



  1.  Coons AH, Creech HJ, Norman Jones R, Berliner E (1942). "The Demonstration of Pneumococcal Antigen in Tissues by the Use of Fluorescent Antibody". The Journal of Immunology. 45 (3): 159–170. 
  1. Cohen, Cynthia. 8/20/2020. “From Stain to Shining Stain: Four decades of progress in immunohistochemistry”. Longer Read. 
  1. Tan WCC, Nerurkar SN, Cai HY, Ng HHM, Wu D, Wee YTF, Lim JCT, Yeong J, Lim TKH. Overview of multiplex immunohistochemistry/immunofluorescence techniques in the era of cancer immunotherapy. Cancer Commun (Lond). 2020 Apr;40(4):135-153. doi: 10.1002/cac2.12023. Epub 2020 Apr 17. PMID: 32301585; PMCID: PMC7170662. 
  1. Hofman P, Badoual C, Henderson F, Berland L, Hamila M, Long-Mira E, Lassalle S, Roussel H, Hofman V, Tartour E, Ilié M. Multiplexed Immunohistochemistry for Molecular and Immune Profiling in Lung Cancer-Just About Ready for Prime-Time? Cancers (Basel). 2019 Feb 27;11(3):283. doi: 10.3390/cancers11030283. PMID: 30818873; PMCID: PMC6468415. 
  1. Giesen, C., Wang, H., Schapiro, D. et al. Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry. Nat Methods 11, 417–422 (2014) doi:10.1038/nmeth.2869

Written by Apple Lewis, BS, MS, HTL(ASCP)