MULTICOLOR BRIGHTFIELD IN-SITU HYBRIDIZATION ON TISSUE-SECTIONS

We describe the brightfield microscopical detection of multiple DNA ta rget sequences in cell and tissue preparations. For this purpose, chro mosome-specific DNA probes labelled with biotin, digoxigenin or fluore scein were simultaneously hybridised and detected by enzyme cytochemis try using two horseradish peroxidase (PO) reactions and one alkaline p hosphatase (APase) reaction. For triple-colour detection on single cel l preparations, the combination of the enzyme precipitates PO/diaminob enzidine (DAB, brown colour), APase/fast red (FR, red colour) and PO/t etramethylbenzidine (TMB, green colour) resulted in an accurate detect ion of DNA targets. Embedding of the preparations in a thin crosslinke d protein layer further stabilised the enzyme reaction products. For i n situ hybridisation on tissue sections, however, this detection proce dure 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 re action was replaced by the APase/new fuchsin (NF, red colour) reaction and the washing steps after the PO/TMB reaction were restricted to th e use of phosphate buffer pH 6.0. Furthermore, to improve the efficien cy of the ISH reaction, APase/NF was applied in an avidin-biotin compl ex detection system and, to avoid target shielding in the triple-targe t ISH, the third primary antibody was applied prior to the second enzy me cytochemical reaction. These adaptations resulted in stable, well c ontrasting brown, red and green coloured precipitates. After quick hae matoxylin counterstaining, the tissue preparations were directly mount ed in phosphate buffer and, optionally, embedded in the crosslinked pr otein layer.