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From tissue fixation to staining: Do you know the mysterious process of immunohistochemistry?
Immunohistochemistry is an immunostaining technique that focuses on the selective recognition of antigens (proteins) in cells and tissues by antibodies. This technique evolved from the immunofluorescence technique originally developed by Albert Hewitt Coons and others in 1941. Over time, immunohistochemistry has become very common in cancer diagnosis and basic research, helping scientists explore the distribution of biomarkers and differentially expressed proteins in different biological tissues.
Sample Preparation
Immunohistochemistry can be performed on tissues that have been fixed and embedded in paraffin, or on frozen tissues. Before sampling, a series of different steps need to be carried out depending on how the tissue was preserved. The general steps include: appropriate fixation, antigen retrieval, incubation with primary antibodies, and subsequent incubation with secondary antibodies.
Tissue Preparation and Fixation
Fixing tissue is critical to maintaining tissue structure and cell shape. The formulation of the fixative, the ratio of fixative to tissue, and the fixation time will significantly affect the final results. 10% neutral buffered formalin is usually used as the fixative, and the fixation time is usually 24 hours at room temperature.
Slice
Tissue samples were sectioned using a microtome. For paraffin-embedded tissue, a thickness of 4 microns is a common standard, while frozen sections are typically 4 to 6 microns thick. The thickness of the sections is critical and different thicknesses may affect the visualization of antigens, so extreme caution must be exercised when performing immunohistochemistry.
Antigen Retrieval
In fixed tissue sections, antigen retrieval makes antigenic sites visible to antibodies. Methane bridges or cross-links of amino groups may form during fixation, which may hinder antibody binding. The most common method of antigen retrieval is to recover latent antigenicity by heating and soaking in buffer.
Tag Sample
Labeling of samples can be accomplished using antibodies labeled with fluorescent compounds, metals, or enzymes to effectively distinguish target antigens.
Detection Methods
The direct method is a single-step staining method, while the indirect method involves an unlabeled primary antibody first binding to the target antigen, followed by the addition of a secondary antibody that binds to the primary antibody. Due to the signal amplification effect of the indirect method, it has higher sensitivity and is widely used in the detection of multiple antigens.
Detection reporter molecules
The reporter molecules detected vary depending on the detection method, the most common ones are chromogen and fluorescent detection. In chromogenic immunohistochemistry, antibodies are usually conjugated to an enzyme and produce a visible color in the presence of a chromogenic substrate. In immunofluorescence detection, the antibody is labeled with a fluorophore.
Application in clinical practice
Immunohistochemistry techniques have played a huge role in diagnostic surgical pathology, especially in the immunophenotyping of tumors (e.g., identifying markers for breast cancer). It has a wide range of applications, including neuroscience and tumor diagnostics, helping researchers explore how proteins behave in specific tissues.
Facing the Challenge
In immunohistochemistry, there are several steps that can lead to different problems, such as excessive background staining or insufficient antigen labeling.
To solve these problems, researchers need to optimize antibody quality and technology.
ConclusionImmunohistochemistry is not only an effective detection technique, it also allows us to discover and understand the distribution of proteins in biological tissues. As technology advances, its application in clinical and research fields will continue to expand. Are you willing to explore the new horizons this technology may open up?
