METHODS FOR SIMULTANEOUS INTERPHASE IN-SITU HYBRIDIZATION AND NUCLEARANTIGEN IMMUNOCYTOCHEMISTRY IN T47-D CELLS

Procedures that combine immunocytochemistry (ICC) and in situ hybridiz ation (ISH) techniques are now used to investigate phenotype/genotype relationships in the same cells. In this report we describe three rapi d procedures for simultaneous detection of a nuclear antigen, progeste rone receptors (PR), and the centromeric region of chromosome 11 (to w hich the human PR gene has been assigned) in T47-D cells. Proteins wer e stained by precipitates of horseradish peroxidase-diaminobenzidine ( PO-DAB, brown color), alkaline phosphatase-Fast Red (APase-Fast Red, r ed color) or alkaline phosphatase-nitroblue tetrazolium-X-phosphate (A Pase-NBT-X-Phosphate, blue color) respectively. To obtain a suitable c ontrast for the two labels, we detected DNA on PO-DAB and APase-NBT-X- phosphate-immunostained cells with interphasic fluorescent in situ hyb ridization (FISH). By contrast, we combined the APase-Fast Red ICC wit h 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 num ber of chromosome 11. This method easily provides simultaneous localiz ation of DNA and protein targets in the same cells and should be appli cable to many other situations.