However, developments in automation have led to a much more efficient workflow, providing high-throughput sample preparation and staining.
These high-throughput devices are called automated immunohistochemistry stainers, IHC stainers, autostainers, or automated slide stainers, and provide greatly increased turnaround times. They come in various formats, from compact benchtop models to larger fully open systems.
IHC staining is critical to several applications and plays an important role in pathology and histology by detecting the presence of specific protein markers. These markers use antibodies that bind with specific antigens to help accurately classify and diagnose abnormal cells, such as those found in cancerous tumors. Antigens are a type of protein found throughout certain cells.
The diagnosis these markers provide can be used to determine whether or not a cancerous tumor is benign or malignant, as well as the cancer’s severity, among other medical analyses.
IHC can also be used for basic research, including the study of biomarker distribution and localization and distinctly expressed proteins in different parts of the body, along with normal tissue and organ development, pathological processes, and cell death and repair.
Furthermore, IHC staining has become a complementary technique to H&E staining, short for hematoxylin and eosin, and Special Stain techniques.
While H&E and Special Stain are non-specific, IHC provides the ability to stain specific protein markers. You will also see scientists and researchers combine in-situ hybridization (ISH) methods with immunostaining to generate more complete pictures of protein and mRNA expression in specific tissue sections and cell types.
ISH is a unique molecular analysis method because it provides the precise microscopic localization of DNA, mRNA and microRNA.
There are different approaches and variations in immunohistochemistry, however, the main preparative steps are as follows:
A tissue sample must be obtained for the diagnosis from a patient. Upon excising, this sample must undergo a process of fixation. This is done using either physical or chemical methods. Depending on the type of tissue being studied, a specific method is generally preferred. Fixation is important because it maintains the tissue structure and ensures antigenicity, the availability of antigens to be detected.
One example of fixation includes the formalin-fixed, paraffin-embedded method of preserving a specimen, which is also referred to as FFPE. A sample is immediately immersed in formaldehyde, which maintains the tissue’s structure and antigens, and is then dehydrated so that it can be embedded into a paraffin wax block.
This makes extremely thin slicing of the sample much easier, as the slices will have to be mounted onto a microscopic slide for examination.
Chemical modification, such as the FFPE method, can make the detectability of antigens or proteins in a tissue sample difficult. Antigen retrieval agents can be used to help improve antigen expression, by eliminating or reducing the effects of chemical treatment.
By breaking down formalin-induced antigen cross-linking, the epitopes on an antigen are re-exposed for binding. An epitope is the part of an antigen molecule that binds to an antibody. Antibodies are used to attach to a specific target antigen. By re-exposing the antigens, primary and secondary antibodies, such as polyclonal and monoclonal, can more easily bind with the sample, effectively staining target antigens for detection.
The two main methods of antigen retrieval are heat and enzyme retrieval, or heat-induced epitope retrieval (HIER), and protease-induced epitope retrieval (PIER) respectively.
Preventing non-specific binding of antibodies and reagents to the tissue of a sample being used for IHC is essential. Even if an antibody has high specificity towards a target antigen, it is possible for non-specific binding to occur. This includes antibodies adsorbing to different types of surfaces, charge-based binding with proteins, and hydrophobic interactions, which all effectively prevent accurate visualization of the targeted antigen in the tissue sample.
Blocking is often the last preparative step performed on the tissue sample before labeling and visualizing. After the sample has been treated appropriately, it is incubated in a blocking buffer for a predetermined amount of time, which may be anywhere from 30 minutes to overnight, either at ambient room temperature or in a refrigerator kept at 4°C.
Washing of the sample after incubation can happen but is not necessarily required, as it may dilute the primary antibodies. Some common types of buffer include conventional serum, protein solutions like sera and bovine serum albumin (BSA), and pre-formulated commercial buffers that contain highly purified single proteins or proprietary protein-free compounds.
Once the tissue sample has been correctly prepared, staining can occur. This can be accomplished using two different methods, an indirect assay or direct labeling. An indirect assay, also referred to as indirect labeling, requires two main steps:
This method is considered more sensitive due to its use of secondary antibodies, but requires more time, and the washing steps involved may affect the amplification of the staining. Direct labeling is quicker than indirect labeling, as it only requires one antibody for staining. This method involves the target antigen reacting directly with the labeled primary antibody, removing the need for multiple incubation and washing steps.
Although simple and rapid, direct labeling is much less sensitive and produces a lower signal amplification in contrast to indirect labeling.
IHC staining has been used as a diagnostic tool for quite some time, but has more recently begun to become automated. The laborious manual preparations involving IHC staining are being replaced with automation in order to diminish the negative effects on efficiency in many histology and medical labs.
Automation of several steps has made the lab technician’s job increasingly manageable when facing a shortage in staff, and has reduced the requirement for intensive training in IHC, effectively spreading the workload between a larger portion of the available staff.
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