Immunocytochemistry
Immunocytochemistry (ICC) is a common
Immunocytochemistry vs. immunohistochemistry
Immunocytochemistry differs from
There are many ways to prepare cell samples for immunocytochemical analysis. Each method has its own strengths and unique characteristics so the right method can be chosen for the desired sample and outcome.
Cells to be stained can be attached to a solid support to allow easy handling in subsequent procedures. This can be achieved by several methods: adherent cells may be grown on microscope slides, coverslips, or an optically suitable plastic support. Suspension cells can be centrifuged onto glass slides (
Concentrated cellular suspensions that exist in a low-viscosity medium make good candidates for smear preparations. Dilute cell suspensions existing in a dilute medium are best suited for the preparation of cytospins through cytocentrifugation. Cell suspensions that exist in a high-viscosity medium, are best suited to be tested as swab preparations. The constant among these preparations is that the whole cell is present on the slide surface. For any intercellular reaction to take place, immunoglobulin must first traverse the cell membrane that is intact in these preparations. Reactions taking place in the nucleus can be more difficult, and the extracellular fluids can create unique obstacles in the performance of immunocytochemistry. In this situation, permeabilizing cells using detergent (Triton X-100 or Tween-20) or choosing organic fixatives (acetone, methanol, or ethanol) becomes necessary.
Antibodies are an important tool for demonstrating both the presence and the subcellular localization of an antigen. Cell staining is a very versatile technique and, if the antigen is highly localized, can detect as few as a thousand antigen molecules in a cell. In some circumstances, cell staining may also be used to determine the approximate concentration of an antigen, especially by an image analyzer.
Methods
There are many methods to obtain immunological detection on tissues, including those tied directly to primary antibodies or antisera. A direct method involves the use of a detectable tag (e.g., fluorescent molecule, gold particles, etc., ) directly to the antibody[3] that is then allowed to bind to the antigen (e.g., protein) in a cell.
Alternatively, there are many indirect methods. In one such method, the antigen is bound by a primary antibody which is then amplified by use of a
Some examples of substrates used (also known as chromogens) are AEC (3-Amino-9-EthylCarbazole), or DAB (
Alternatively the secondary antibody may be covalently linked to a fluorophore (FITC and Rhodamine are the most common) which is detected in a fluorescence or confocal microscope. The location of fluorescence will vary according to the target molecule, external for membrane proteins, and internal for cytoplasmic proteins. In this way immunofluorescence is a powerful technique when combined with confocal microscopy for studying the location of proteins and dynamic processes (exocytosis, endocytosis, etc.).
References
- ISBN 978-1-4419-1304-3.
- ^ Renshaw, Simon (2017). Immunohistochemistry and Immunocytochemistry: Essential Methods, Second Edition. John Wiley & Sons, Ltd. pp. 35–102.
- ^ Cooper, Mark; Lummas, Sheriden (2016). Immunohistochemistry and Immunocytochemistry: Essential Methods, Second Edition. John Wiley & Sons, Ltd. pp. 21–23.