33.5:
Immunocytochemistry and Immunohistochemistry
Immunocytochemistry or ICC and immunohistochemistry or IHC are staining techniques that use enzyme-linked antibodies to visualize and quantify specific proteins or antigens in cultured cells or tissue sections.
In ICC, a single layer of targeted cells is grown on a glass coverslip. The cells are fixed with a cross-linking agent to prevent enzymatic degradation of the antigen, then treated with a detergent to make the cell membrane permeable.
In IHC the whole tissue is fixed and embedded in a block of paraffin.Then, the sample is sectioned into thin slices, which can be placed on slides. Finally, the paraffin is removed to retrieve the antigen.
Once the antigens are accessible in the sample, they are bound by target-specific primary antibodies linked with conjugate enzymes like horseradish peroxidase.
The enzyme catalyzes the oxidation of DAB stain to a brown precipitate allowing visualization of the protein of interest.
If the signal detected is weak, secondary antibodies with conjugate enzymes are used to bind an unlabeled primary antibody on the target protein for further signal amplification.
33.5:
Immunocytochemistry and Immunohistochemistry
Immunocytochemistry (ICC) and immunohistochemistry (IHC) are techniques that use antibodies to check for specific proteins or antigens in a sample. The technique was first published by Albert Coons in 1941 to detect the presence of pneumococcal antigen in tissue sections from mice infected with Pneumococcus. Immunocytochemistry helps localization of proteins or antigens in individual cells like blood cells, stem cells, etc., while immunohistochemistry does the same for tissue samples.
These techniques can be colorimetric if enzyme-conjugated antibodies are used or if fluorescence-based fluorophore-tagged antibodies localize the proteins or antigens of interest. The colored product obtained via the enzymatic reaction or fluorescence emitted by the fluorophore is then visualized under an optical or immunofluorescence microscope.
These techniques can use primary or secondary antibodies tagged with an enzyme or a fluorophore. If a primary antibody is used, the technique is referred to as direct ICC or IHC, as the conjugated antibodies directly bind to the epitope of the protein or antigen of interest. In indirect, a secondary antibody tagged with the enzyme or fluorophore binds to the primary antibody attached to the epitope of the protein or antigen of interest. The signal emitted upon binding of a secondary antibody is stronger than the signal from a primary antibody.
Both the techniques vary slightly in the steps of sample preparation. In ICC, a monoculture layer of cells is generated, which are then treated with fixing agents like formaldehyde to prevent the enzymatic degradation of the antigens, followed by treatment with detergents like Triton X or Tween 20 to make the cell membrane permeable for the entry of antibodies. Similarly, in IHC, the tissue samples are first treated with a fixing solution, followed by embedding in paraffin wax to prevent damage to fragile tissue. The tissue samples are then sectioned into thin slices and treated with detergent before the introduction of antibodies.
Once the cells or tissue slides are treated with detergent, the antibodies are then added. In direct ICC or IHC, the primary antibody tagged with fluorophore or enzymes is added, and post-incubation, they are visualized under immunofluorescence or optical microscope. In indirect ICC or IHC, the samples are first incubated with the primary antibody, followed by washing to remove unbound antibodies. The secondary antibody is then introduced, which binds with the primary antibody and emits fluorescence or colored product upon adding dye. The samples are now ready for visualization.
Suggested Reading
- Skoog, L. and Tani, E., 2011. Immunocytochemistry: an indispensable technique in routine cytology. Cytopathology, 22(4), pp.215-229.
- Duraiyan, J., Govindarajan, R., Kaliyappan, K. and Palanisamy, M., 2012. Applications of immunohistochemistry. Journal of pharmacy & bioallied sciences, 4(Suppl 2), p.S307.