Cytochemical detection systems for in situ hybridization, and the combination with immunocytochemistry, 'who is still afraid of red, green and blue?'.

Abstract

An overview is given of the different non-radioactive cytochemical detection methodologies that are currently utilized to localize nucleic acid sequences in chromosomes, cells and tissue sections. Dependent on the reporter molecule (fluorochrome, enzyme or hapten) that is used to modify the appropriate nucleic acid probe, and the sensitivity that is required, the in situ hybridized sequences can be detected either directly after hybridization or indirectly, using cytochemical detection and amplification layers. These may then contain antibody and/or avidin molecules conjugated to fluorochromes, enzymes or colloidial gold particles. Since the choice of a suitable probe-labelling method in combination with a fluorescence, enzyme cytochemical or immunogold-silver detection procedure is often determined by the user's own practical experience and applications, the different detection methodologies are compared with each other in detail with respect to sensitivity, resolution, applicability for multiple probe detection, and signal evaluation. Furthermore, procedures are reviewed for the combination of in situ hybridization with immunocytochemical detection of proteins and/or incorporated bromodeoxyuridine, which allow the simultaneous visualization of genomic phenotypic and/or cell cycle parameters in the same sample. Possible improvements with respect to sensitivity, specificity and multiplicity of the detection methods, which may be interesting for one's own experimental design, are finally being discussed.