#1 Introduction to Immunohistochemistry

#1 Introduction to Immunohistochemistry

Anthony van Leeuwenhoek is credited with being the first person to use a microscope to view tiny “animacules” in 1674. At approximately the same time, Robert Hooke used a similar microscope to view thin slices of cork. The structures that he observed resembled the tiny cells that monks lived in at the local monastery, so he named the structures “cells”.

From that time through the 1800’s and early 1900’s, laboratorians experimented with various dyes to impart contrast to sections on microscope slides in order to see these cells. Joseph von Gerlach is credited with developing the first histology stain by using a solution of carmine in 1858 to stain brain cells. Several years later, in 1896, the hematoxylin and eosin (H&E) stain combination was worked out by Paul Mayer. The H&E stain has since remained the cornerstone of pathological diagnosis to this day.

The mid 1900’s saw the rapid development of many special stains. These stains are also used in present day histology laboratories to detect microorganisms, cell products and tissue structures. Similar to the H&E stain, these special stains use colored dye solutions to impart color and contrast to the desired structures. However, many times cells under study appear normal with these stains, even though they are later proved by other methods to display pathology.

In the 1960’s and 1970’s a novel method was developed which helped pathologists to diagnose cancer cells. The method was based on using antibodies to specifically bind to protein antigens within the cells and tissue elements of a tissue section on a microscope slide. This was followed by using a “detection chemistry” to visualize the binding, such that it could be seen using a light microscope. The method is called immunohistochemistry, or “IHC” for short.

Since that time many thousands of antibodies have been developed for use in IHC. Each one will specifically bind with only one protein site. Through experimentation and research, pathologists now have a large menu of different proteins which can be detected and visualized within the specimens on the microscope slides. The presence or absence of these proteins provides information from which to form a diagnosis.

The IHC method relies on the ability to develop and use antibodies that are directed to, and bind only with, very specific proteins. Early literature described the binding as a “lock and key” arrangement. However, it was soon discovered that the binding of an antibody to its protein antigen counterpart is a very complex, three-dimensional event.
Mammals have an immune system that makes antibodies (Ab) to foreign proteins, for example, bacteria and viruses. If you inject mice, rabbits, or other animal subjects with pure protein preparations, their immune system will make antibodies to the protein. These proteins are also referred to as antigens (Ag). The antibodies are highly specific – each will bind to only one antigen.

For example, if you are exposed to the chicken pox virus, you most likely will contract the chicken pox disease. For 2-4 weeks from the exposure time, your immune system makes antibodies to the chicken pox virus. From that point on, any time you are exposed to the chicken pox virus, the antibodies circulating in your blood protect you from the disease by binding with and inactivating the chicken pox virus particles.

In histology, we can use pure antibody preparations to specifically bind to protein antigens in tissue sections. Since antibodies themselves are too small to be seen under a microscope, we can “label” them with colored tags which can be observed under the microscope. The proteins can be localized within cells, outside of cells, or even within cell nuclei.

Future segments of the educational series will delve into the methodology of this fascinating technique.

Figure 1. IHC staining for the proteins of keratin in a microscopic section of skin. Keratin is a protein that is produced by epidermal cells and in this preparation is stained brown. Cell nuclei are visualized by a blue hematoxylin counterstain. Original magnification X 400.


Tell us which IHC techniques work in your laboratory.

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1. Chapman C.M. (2017). The Histology Handbook.  Amazon CreateSpace Independent Publishing Platform
2. Chapman C.M., Dimenstein I.B. (2016).  Dermatopathology Laboratory Techniques.  Amazon CreateSpace Independent Publishing Platform
3. https://www.nature.com/milestones/milelight/full/milelight02.html
4. Von Gerlach, J. (1858). Mikroskopische Studien aus dem Gebiet der menschlichen Morphologie (Enke)
5. 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

Immunohistochemistry (IHC) – Basics

Immunohistochemistry (IHC) – Basics

There are some pathological conditions that exist that cannot be accurately diagnosed by examining hematoxylin and eosin (H&E) stained slides alone. In such cases, the pathologist may order immunohistochemical (IHC) stains to help render a diagnosis. Immunohistochemical stains are classified as either immunofluorescence (IF) or immunoperoxidase; however both make use of highly specific antibody preparations to detect their specific antigens in tissue sections. Direct immunofluorescence (DIF) utilizes a fluorescent label attached directly to the antibody. Upon viewing under ultraviolet (UV) light in a dark field microscope, the label fluoresces an apple green color (Figure 1).

Immunofluorescence is further classified into direct immunofluorescence (DIF) or indirect immunofluorescence (IIF). This procedure is done for patients who present with bullous (i.e. blistering) disease, which may have an autoimmune cause. DIF is done by cutting frozen sections of the patient’s skin specimen, and then incubating these slides with specific fluorescein labeled antibodies. IIF makes use of the patient’s serum, serially diluted and incubated on substrate slides (i.e. frozen sections of monkey esophagus). A second fluorescein labeled antibody is then applied. The resulting slides of both DIF and IIF procedures are then viewed in a dark field fluorescence microscope and the pathologist records the resulting staining patterns to make an accurate diagnosis Figure 2).

Immunoperoxidase stains are performed on sections of formalin fixed paraffin embedded tissue (i.e. the identical material from which the H&E slide is made from). Sections are cut, baked and then hydrated to stain either manually or with automated technology. The resulting specific chromogen stain can be viewed under regular light microscopy. Whether or not a particular antigen is present helps the pathologist to make an accurate diagnosis.

Immunoperoxidase (IP) methods are based on the same mechanism as IF methods. Specific antibodies are used to detect and bind to specific antigens located within the tissue section. These antigens may be proteins located on the exterior of the cell, within the interior of the cell, on the cell membrane, on the nuclear membrane, or within the nucleus. [There are even other, in situ methods, that can detect gene aberrations within the nucleus. We will explore these methods in a future blog.]

Antibodies themselves are too small to be seen under a microscope. So, like the IF procedures, the IP procedures employ methods to “label” the site of antibody: antigen binding. Figure 3 is a schematic representation of the IP method. The primary antibody binds to the antigen present in the tissue section. A secondary antibody, labeled with a link (either a biotin or polymer link) is applied, and binds to the primary antibody. The final detection molecules are added, and bind to the link reagents. These detection molecules form colored precipitates at the site of antibody binding when they are developed. Final reaction products can be brown, red or blue, depending upon the chromogen used (Figure 4).

An important detail is that specimens for DIF must be collected into a specific immunofluorescence transport medium, such as Michel’s medium, in the clinicians’ office. The specimens must then be recognized when they arrive in the histology laboratory and routed to the immunohistochemistry laboratory. At no time can these specimens be exposed to formaldehyde. Formaldehyde exposure renders these specimens unsuitable for immunofluorescence.

Detection of protein antigens within tissues and cells assists the pathologist in making accurate diagnoses. This is especially important when the diagnosis cannot be made entirely with the H&E slide. The results may also be used in determining how to treat the patient.






Theory and Practice of Histological Techniques. JD Bancroft, A Stevens ed. Churchill Livingstone, NY. Fourth edition. 1996
Theory and Practice of Histotechnology. DC Sheehan, BB Hrapchak. CV Mosby Company, St. Louis. First edition. 1980.