Methods for simultaneous interphase in situ hybridization and nuclear antigen immunocytochemistry in T47-D cells

Procedures that combine immunocytochemistry (ICC) and in situ hybridization (ISH) techniques are now used to investigate phenotype/genotype relationships in the same cells. In this report we describe three rapid procedures for simultaneous detection of a nuclear antigen, progesterone receptors (PR), and the centromeric region of chromosome 11 (to which the human PR gene has been assigned) in T47-D cells. Proteins were stained by precipitates of horseradish peroxidase-diaminobenzidine (PO-DAB, brown color), alkaline phosphatase-Fast Red (APase-Fast Red, red color) or alkaline phosphatase-nitroblue tetrazolium-X-phosphate (APase-NBT-X-Phosphate, blue color) respectively. To obtain a suitable contrast for the two labels, we detected DNA on PO-DAB and APase-NBT-X-phosphate-immunostained cells with interphasic fluorescent in situ hybridization (FISH). By contrast, we combined the APase-Fast Red ICC with an immunocytochemical ISH using alkaline phosphatase-NBT-X-phosphate detection. Only the procedure combining APase-NBT-X-phosphate ICC and FISH ensures optimal visualization of both the PR content and the number of chromosome 11. This method easily provides simultaneous localization of DNA and protein targets in the same cells and should be applicable to many other situations.