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Multicolour preparations for in situ hybridization using precipitating enzyme cytochemistry in combination with reflection contrast microscopy

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Abstract

We have further developed a method for the detection of different enzyme cytochemical reaction products by means of reflection contrast microscopy (RCM). By embedding these enzyme precipitates in a protein matrix, we were able to prevent the reaction products from dissolving in immersion oil, which is required for RCM analysis. The applicability of the RCM procedure is, therefore, extended to a range of cytochemical enzyme precipitation methods, which normally result in oil soluble reaction products. To test their usefulness, these enzyme precipitates have been used in single- as well as double-label in situ hybridization (ISH) procedures to visualize a number of DNA target sequences by several different reflection colours, i.e. white, yellow and red. Three repetitive DNA probes for the (sub)centromeric regions of chromosomes 1, 7 and 17, as well as a repetitive DNA probe for the telomeric region of chromosome 1, and two cosmid DNA probes (40 kb each) for both arms of chromosome 11 could be detected with high efficiency in both interphase and metaphase preparations. Moreover the enzyme precipitates were shown to be stable upon exposure to excitation light or upon storage. It may be concluded that these findings render RCM a sensitive method for the visualization of multiple targets in biological specimens.

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Authors and Affiliations

  1. Department of Molecular Cell Biology & Genetics, Cardiovascular Research Institute, University of Limburg, Maastricht, The Netherlands

    Ernst J. M. Speel, Miriam Kamps, Frans C. S. Ramaekers & Anton H. N. Hopman

  2. Department of Cytochemistry and Cytometry, University of Leiden, Leiden, The Netherlands

    Jan Bonnet

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  1. Ernst J. M. Speel
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  2. Miriam Kamps
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  3. Jan Bonnet
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  4. Frans C. S. Ramaekers
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  5. Anton H. N. Hopman
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Speel, E.J.M., Kamps, M., Bonnet, J. et al. Multicolour preparations for in situ hybridization using precipitating enzyme cytochemistry in combination with reflection contrast microscopy. Histochemistry 100, 357–366 (1993). https://doi.org/10.1007/BF00268934

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