Immunostaining has diverse applications. It can be used to confirm the presence of particular antigens, quantify levels of a protein and locate cellular protein and components with greater precision than standard histological staining techniques. Antibodies are utilized, which are raised to target antigens on a protein or other macromolecule with an inherently high binding affinity. It is routinely used to diagnose disease indicate the presence of certain hormones as in pregnancy tests (Jeyapradha et al., 2012). It can also be used to distinguish cell types and the stage of cell cycle as distinct proteins are expressed in each case and provide unique antigen targets for antibodies (Yanagita et al., 2012).

Primary antibodies are raised to directly bind to the antigen of interest in direct immunostaining. Often the signal is weak so amplification is favoured via indirect immunostaining, whereby secondary antibodies are raised against the primary antibody. These can be Polyclonal antibodies, which bind to multiple different epitopes on the primary antibody. Thus, a single primary antibody may have several different epitopes, each of which can have a distinct secondary antibody attached, hence the signal is multiplied proportionately.

A range of signal molecule types are available for antibody conjugation. These are linked to the antibody via covalent bonds and emit a signal to be detected or visualized, thereby indicating the presence of target antigen at the binding site.

Immunofluorescence uses fluorochromes as signalling molecules. These emit visible light under ultraviolet light excitation which is observed under fluorescence microscope to visualize targeted proteins and their localities in tissue, cells or membrane blots. Different coloured fluorochromes are used to identify different antigens for a more complete visualization of cellular protein distribution. Indirect fluorescence with secondary antibodies, is generally employed to multiply the primary signal sufficiently. The drawback with immunofluorescence is impermanence and fading of fluorescence, making samples poor archival material. Also UV microscopes are less practical than light microscopes. Therefore, immunofluorescence is largely superseded by enzymatic methods (Mölne et al, 2005).

Enzyme signalling molecules such as Horseradish peroxidase or alkaline phosphatase can be conjugated to antibodies. These catalyse the breakdown of an array of possible substrates, each of which generates a distinctly coloured by-product, allowing different proteins of interest to be highlighted in different colours for protein mapping in tissues and cells. An advantage of this technique is that standard staining methods can be used in conjunction to more completely visualize cellular architecture and the relative localities of targeted proteins (Mölne et al, 2005).

Antibodies may also be conjugated to biotin which has ultra-high binding affinity for certain proteins, which minimizes background staining and up to 6 biotin molecules can be joined to a single antibody, eliciting inherent amplification (Hsu, S.M. 1981). Furthermore, other signal molecules can be simultaneously conjugated to the antibody for an amplified signal.

Radioactive compounds can also be conjugated to antibodies and the radiotracer emissions detected via autoradiography. This is an extremely sensitive assay and is useful for quantifying protein concentrations.

Bromodeoxyuridine labelling is a method to detect proliferating cells. brdU is an artificial analogue of thymidine and gets incorporated into DNA during mitosis. Anti-brdU antibodies bind to the brdU incorporated DNA, which makes this technique particularly useful for identifying rapidly dividing cancerous cells.

Positive and negative controls should be employed to eliminate false positive results. For negative controls, primary antibody is omitted and the sample flooded with animal serum from which secondary antibodies were raised. The secondary antibodies should not bind and simply wash away so no staining occurs. Positive controls typically employ a solution or tissue sample known to contain the antigen of interest so antibody-antigen binding is certain and this validates the immunostaining method.

Antigen retrieval re-exposes antigen binding sites where tissue fixing and processing caused epitope masking. Various methods are employed such as heat induced epitope retrieval where the sample is incubated in heated buffer solution. Protease induced epitope retrieval is an alternate method in which the sample is incubated with a protease. Non-specific binding measures should be taken to inhibit antibodies from binding at undesired sites which would generate false positive results. Adding serum to saturate the non-target antigens with serum protein is a recommended procedure. Milk can even be used for a Western Blot.

The degree of specificity of immunostaining makes it an invaluable, relatively accurate tool for protein detection, visualization, quantification and disease diagnosis. Using antibodies enables signal amplification as well as high binding affinity and specify to precise 3D epitope structures or amino acid sequences which are typically 6 amino acids in length but can be a single amino acid with side chain (Hebbes et al, 1989).

 

 

Jeyapradha, D.,& Rajeshwar, G., & Karunakaran, K., & Murugesan, P.  (2012) Applications of immunohistochemistry. J Pharm Bioallied Sci.: S307–S309.

Yanagita, E., Kamoshida, S., Imagawa, N., Itoh, T. (2012) Immunohistochemistry-based cell cycle detection (iCCD): a novel system to visualize cell kinetics on formalin-fixed paraffin-embedded tissues.Am J Surg Pathol. 769-73

Mölne, J., Breimer, M.E., Svalander, C.T. (2005) Immunoperoxidase versus immunofluorescence in the assessment of human renal biopsies. Am J Kidney Dis. 674-83.

Hsu, S.M., Raine, L. J (1981) Protein A, avidin, and biotin in immunohistochemistry. Histochem Cytochem. 1349-53.

Hebbes T.R., Turner C.H., Thorne A.W., Crane-Robinson C. (1989) A “minimal epitope” anti-protein antibody that recognises a single modified amino acid. Mol Immunol.865-73.