IHC Antibodies

Tissue Preparation

The tissue sample preparation is of crucial importance, because inadequate handling may disrupt tissue structure, cause reduced antibody binding affinity or even prevent binding altogether. The goal is to preserve the tissue in close to life-like conditions while concomitantly preventing autolysis and degradation due to bacterial or fungal growth. To this end, fixatives are used, such as paraformaldehyde-lysin-periodate (PLP) or formalin. The most common fixative is 4%-10% (in some cases up to 40%) formaldehyde in 0.1M phosphate buffer (in order to stabilize the pH).

For tissue that does not tolerate formaldehyde incubation, an often used alternative is that samples are snap-frozen in liquid nitrogen, and then cut into sections. Tissues incubated with fixatives are embedded in a matrix, e.g. paraffin, or, after they have been dehydrated, in alcohol (isopropanol or ethanol), and then cut into sections. For sensitive tissues the application of a vibratome microtome may be advisable, due to the lower physical stress it produces on the tissues.

Antigen Retrieval

Despite its superior preservation quality, in terms of morphology, formalin-fixed paraffin-embedded samples can lose some or all immunoreactivity due to structural changes of the targeted antigen. The demonstration of antigens, and hence the immunoreactivity, can be improved using various techniques. Mainly, there are two distinct approaches, i.e. heat treatment, the so-called "heat induced epitope retrieval" (HIER), and the "proteolytic induced epitope retrieval" (PIER). In order to apply these techniques tissue-sections have to be deparaffinized and rehydrated first.

HIER works by applying, what is often referred to as retrieval solution. This preheated buffer has varying composition and pH, often consisting of Tris-HCl or citrate. The emerged samples are exposed to heat for varying times (usually between 10-60 minutes) and then slowly cooled. The heating itself may be carried out using an autoclave, waterbath, pressure-cooker or similar appliance.

PIER works by applying digestion enzymes, such as proteinase K, trypsin, pepsin and various other proteinases. Optimal incubation time and concentrations have to be tested. In the final run sample specificities should be optimized. Occasionally it may be required to combine HIER and PIER in order to achieve antigen "unmasking" following formalin-incubation.

Staining Methodologies

Direct method

In this method a labelled antibody (e.g. with a substrate-chromogen) reacts directly with the antigen in the tissue. The advantage is that only one antibody is needed, hence the application is fast and produces little nonspecific binding. On the other hand, since only one antibody binds one epitope, the signal intensity is low. For applications with small amounts of antigen the signal may be too weak.

Indirect method (two-step)

In order to upscale the low signal intensity of the direct method, the indirect method has been developed. The primary antibody binds to the antigen, followed by a second (labelled) antibody binding the primary. The signal is amplified due to the binding of multiple secondary antibodies to a single primary antibody. Another advantage is that only one labelled antibody needs to be employed for different targets, which may reduce costs. One disadvantage is that nonspecific binding occurs more frequently than with the direct method.

Three-step method

In order to further amplify the signal an additional antibody may be employed that binds to the secondary antibody. This method leads to a third layer of antibodies all of which are labelled. This application is useful for staining of antigens with a limited number of epitopes.

PAP method

Today, the peroxidase anti-peroxidase method is rarely used, but has been popular in pathology laboratories previously. It is an indirect method which depends on a third layer of antibodies, bound to peroxidase, that binds an unconjugated second layer antibody. The peroxidase is not chemically conjugated to the IgG but immunologically bound. That means that the third layer antibody is specific for peroxidase. This leads to a much higher activity of the peroxidase, in turn increasing the assay sensitivity by two to three orders of a magnitude.

(Strept)Avidin-Biotin Complex (ABC) methods

Nowadays, one of the most commonly used method for staining is based on the high affinity that avidin (chicken egg) and streptavidin (Streptomyces avidinii) have for the glycoprotein biotin. The basic principle is that avidin (or streptavidin) reacts with a biotinylated secondary antibody, followed by a horseradish peroxidase reaction.

In some cases catalyzed signal amplification (CSA) is employed. An amplification reagent leads to the aggregation of a large number of biotins that subsequently react with streptavidin-labelled peroxidase. One issue with avidin is its large electrostatic binding, due to its molecular charge. Streptavidin does not have the same propensity, thus reducing the background signal.

Polymeric methods

In short, polymeric methods employ large antibody-bound polymers with the ability to bind a larger number of molecules, typically an average of ten antibodies and approximately 70 enzyme molecules. This setup allows for outstanding amplification of the signal, hence high sensitivity, reduced nonspecific binding, and therefore low background signal. It also allows for staining of two different antigens at the same time.