Immunohistochemistry
Antibody Search and Immunohistochemistry
Traditional immunohistochemistry (IHC) techniques rely on enzyme-conjugated antibodies to convert a chromogen substrate to a colorful precipitate. Nowadays, fluorescence detection is as it provides several advantages over traditional chromogenic detection:
- Higher contrast and visibility
- Colocalization studies
- Multiplex analysis
- Large selection of fluorophore-conjugated antibodies
Fluorochrome combinations must be carefully selected to limit emission spectra overlap. Poorly optimized fluorochrome combinations may obscure detection of individual proteins, especially the detection of co-localized proteins. Compatibility with the laser settings of the microscope must also be considered. FF spectra viewer is a platform for the optimal design of the multiplex fluorescence analysis. It allows the rapid visualization of the emission spectrum of more than 1000 fluorophores from all suppliers. Emission and excitation wavelengths and selection of instrument-specific laser settings can also be displayed.
Applications of IHC/ICC:
- Cancer Diagnosis and prognosis
- Cancer progression evaluation
- Identification of infectious agents
- Neurodegenerative disease
- Muscular dystrophy
Immunohistochemistry
Immunohistochemistry is a technique used to identify and visualize the expression and localization of specific antigens using antibodies. IHC is an important tool applied in life-science research and clinical diagnostic to characterize biological processes and disease status and progression.
Immunohistochemistry and immunocytochemistry (ICC) share many similarities from a technical standpoint and the terms are often used interchangeably. The main differences between them are that the first uses tissue sections whereas the second uses cells. The general protocol for IHC and ICC is basically the same.
IHC/ICC workflow
Sample preparation
There are different ways to prepare and preserve samples for IHC and ICC. Each has different effects on epitope availability and tissue/cell structure preservation. For example, epitope preservation is optimal in frozen tissue but tissue/cell structure is better preserved in formalin-fixed paraffin-embedded (FFPE) sections.
Fixation
Preserves cellular components, preventing autolysis and displacement of cell constituents, and facilitates staining.
Antigen retrieval
This is necessary when cross-linking reagents are used for fixing. The methylene bridges formed between proteins’ amino groups alter their 3D conformation affecting antibody recognition and binding.
Antigen-antibody reactions
When selecting a specific antibody against the target antigen it is important to consider how the sample was prepared and whether the resulting epitope retained its conformation during the process. Antibody clonality and detection methods are also factors that must be kept into account.
