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.
