Prior to IHC, tissues are removed from the patient or animal and either frozen or chemically preserved (fixed) and then embedded in paraffin wax. Sections as thin as 4 μm are sliced from frozen or paraffin-embedded tissue and mounted onto slides in preparation for antibody-mediated staining. In this way, researchers can look at the localization of the target antigens within cellular components while maintaining the original architecture of the surrounding tissue, as shown in the right panel below.
For ICC, most, if not all, of the extracellular matrix and other stromal components are removed, leaving only whole cells, as shown in the left panel below. Sample sources for ICC can be from any suspension of cells, obtained from patients or animals (e.g., blood smears, swabs and aspirates) or cultured cells grown in monolayers, usually on sterile glass coverslips. The latter is the most common sample type used for ICC. As mentioned above, ICC is also synonymous with IF staining, especially when we present results with cultured cells. The 2 abbreviations are often used interchangeably in this context.
Besides the biological source, IHC and ICC differ in the amount of sample processing required before antibody-mediated staining. For example, ICC is associated with whole cells that have to be permeabilized, either through a fixation procedure or a separate permeabilization step, to facilitate antibody penetration to the intracellular targets. Depending on the thickness of the section and the method of fixation, IHC samples may not have to undergo a separate permeabilization step. However, most formalin-fixed, paraffin-embedded (FFPE) IHC sections must be further processed prior to antibody staining to uncover latent epitopes on antigenic targets. This is usually referred to as epitope or antigen retrieval.
Once the samples are processed, there are few differences in the antibody staining protocol between IHC and ICC, although, as with all antibody-based staining, the protocol must be optimized based on the nature of the samples to be stained and the antibodies used.
- Coons, A.A., et al. (1942) J Immunol 45, 159-170.
- Hassel, J. and Hand, A.R. (1974). J. Histochem. Cytochem. 22 229-239.
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