Immunohistochemistry (IHC) Services
Immunohistochemistry (IHC) services involve microscopic localization of antigens in cells of a tissue section by staining with antibodies labeled with fluorescent or pigmented reporters. Fluorescent dyes or enzymes, especially horseradish peroxidase, are commonly employed to highlight antibody binding to the target antigen.
Fixation: Tissue preparation is the cornerstone of our immunohistochemistry service. To ensure the preservation of tissue architecture and cell morphology, prompt and adequate fixation is essential, however, inappropriate or prolonged fixation may significantly diminish the antibody’s binding capability. The goal is to preserve the tissue in simulated life-like conditions, while concomitantly preventing autolysis and degradation due to bacterial or fungal growth.
To this end, fixatives are used, such as paraformaldehyde-lysine-periodate (PLP) or formalin. The most common fixative is 4%-10% (but 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 alternative is that samples are snap-frozen in liquid nitrogen, and then cut into sections. Tissues incubated with fixatives are embedded in a matrix. For sensitive tissues the application of a vibratome microtome may be advisable due to the lower physical stress it subjects the tissues to.
ANTIGEN RETRIEVAL: Despite its superior preservation quality, in terms of morphology, formalin-fixed paraffin-embedded (FFPE) samples can mask immunoreactivity due to structural changes of the targeted antigen. The demonstration of antigens, and hence the immunoreactivity, can be improved using various techniques. There are two primary distinct approaches: “heat-induced epitope retrieval” (HIER), and “proteolysis-induced epitope retrieval” (PIER). In order to apply these techniques, tissue-sections must be deparaffinized and rehydrated first.
HIER works by applying what is often referred to as a retrieval solution. This preheated buffer has varying composition and pH but often consists of Tris-HCl or citrate. The submerged samples are exposed to heat for varying periods of time (usually between 10-60 minutes) and then slowly cooled. The heating itself may be carried out using an autoclave, water bath, pressure-cooker or similar appliance. PIER works by applying proteolytic enzymes such as proteinase K, trypsin, pepsin and various other proteinases. Optimal incubation time and concentrations must be tested empirically. 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.
IHC STAINING METHODOLOGIES:
In this method an enzyme-labeled antibody (e.g. in combination 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 often low. For applications involving small amounts of antigen, the signal may be too weak.
Indirect method (two-step)
In order to amplify 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 (labeled) antibody binding to the primary antibody. The signal is amplified due to the binding of multiple secondary antibodies to a single primary antibody. Another advantage is that only one labeled antibody needs to be employed for different targets, which can reduce overall cost. One disadvantage of the approach is that nonspecific binding can occur more frequently than with the direct method.
In order to further amplify the signal an additional antibody may be employed which binds to the secondary antibody. This method leads to a third layer of antibodies, all of which are labeled. This application is useful for staining antigens with a limited number of epitopes.
Today, the peroxidase anti-peroxidase method is rarely used, but has historically been popular in many pathology laboratories. It is an indirect method depending 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 subsequently increasing the assay sensitivity by two to three orders of a magnitude.
(Strept)Avidin-Biotin Complex (ABC) methods
One of the most commonly used methods for staining is based on the high affinity 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 biotin molecules that subsequently react with streptavidin-labeled peroxidase. One issue with avidin is its high electrostatic binding, due to its molecular charge. Streptavidin does not have the same propensity, thus reducing the background signal.
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 the high sensitivity, reduced nonspecific binding, and therefore low background signal. It also allows the staining of two different antigens at the same time.
Karebay™ Biochem provides high quality IHC services and immunohistochemical staining to pharmaceutical, medical device and academic clients. Our team works carefully to standardize immunohistochemistry processes for down-stream whole slide image quantitation. We have industry leading expertise in the use of dual staining in brightfield immunohistochemistry, and have developed a large number of protocols. By choosing KareBay you will receive the best results for your immunohistochemistry needs.
How to get started
Please contact us with your ihc service requests at firstname.lastname@example.org and we will reply with a detailed quote as soon as possible. This process usually takes between 24 and 48 hours and the quote will include an estimated price as well as the time required to complete the project. All inquiries and subsequent projects are handled in strict confidence and will be backed by a confidentiality agreement if desired.