Multi-colour brightfield in situ hybridisation on tissue sections

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 crosslinked 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 crosslinked protein layer.