Multi-colour brightfield in situ hybridisation on tissue sections |
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Authors: | A H N Hopman Sandra Claessen Ernst J M Speel |
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Institution: | (1) Department of Molecular Cell Biology and Genetics, University Maastricht, P.O. Box 616, NL-6200 MD Maastricht, The Netherlands Tel. +31–43–3881358; fax +31–43–3670948 e-mail Hopman@molcelb.unimaas.nl, NL |
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Abstract: | We describe the brightfield microscopical detection of multiple DNA target sequences in cell and tissue preparations. For
this purpose, chromosome-specific DNA probes labelled with biotin, digoxigenin or fluorescein were simultaneously hybridised
and detected by enzyme cytochemistry using two horseradish peroxidase (PO) reactions and one alkaline phosphatase (APase)
reaction. For triple-colour detection on single cell preparations, the combination of the enzyme precipitates PO/diaminobenzidine
(DAB, brown colour), APase/fast red (FR, red colour) and PO/tetramethylbenzidine (TMB, green colour) resulted in an accurate
detection of DNA targets. Embedding of the preparations in a thin cross-linked protein layer further stabilised the enzyme
reaction products. For in situ hybridisation on tissue sections, however, this detection procedure showed some limitations
with respect to both the stability of the APase/FR and PO/TMB precipitates, and the sequence of immunochemical layers in multiple-target
procedures. For this reason, the APase/FR reaction was replaced by the APase/new fuchsin (NF, red colour) reaction and the
washing steps after the PO/TMB reaction were restricted to the use of phosphate buffer pH 6.0. Furthermore, to improve the
efficiency of the ISH reaction, APase/NF was applied in an avidin-biotin complex detection system and, to avoid target shielding
in the triple-target ISH, the third primary antibody was applied prior to the second enzyme cytochemical reaction. These adaptations
resulted in stable, well contrasting brown, red and green coloured precipitates. After quick haematoxylin counterstaining,
the tissue preparations were directly mounted in phosphate buffer and, optionally, embedded in the cross-linked protein layer.
Accepted: 27 June 1997 |
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