Background: Transcriptional activities of genes require intermediary regulators (nuclear factors) that bind to specific segments of nuclear DNA. A method to localize in situ the distribution of these factors using nonradioactive oligonucleotides in paraffin wax-embedded tissues is described. The distribution of two nuclear factors, activated protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB), was studied in two experimental models of immune complex glomerulonephritis in rats and atherosclerosis in rabbits.
Methods: Sections were fixed with 0.2% paraformaldehyde and were digested with pepsin A. Oligonucleotides containing the consensus sequence of NF-kappaB and AP-1 were 3'-labeled with digoxigenin. The preparations were incubated with the labeled probes (4 degreesC, overnight). After washing, the sections were incubated with an antidigoxigenin antibody conjugated with alkaline phosphatase, and the color reaction was developed. In addition, this method was combined with standard immunohistochemistry to identify the cell-type-specific localization of these DNA-binding factors.
Results: Kidney sections from rats with immune complex nephritis showed positive nuclear staining for AP-1 in the nuclei of several glomerular and tubulointerstitial cells. Arteries from rabbits with focal atherosclerosis presented nuclear staining for NF-kappaB in the neointima and media. The nuclear staining was highly specific, as assessed by several negative controls. In addition, Southwestern histochemistry in rabbits, followed by immunohistochemistry, demonstrated that the NF-kappaB activity was present in the area occupied by macrophages and smooth muscle cells.
Conclusion: These results show a novel method of in situ transcription factors detection using nonradioactive probes in paraffin wax-embedded tissues, which allows a simultaneous visualization of the cell-type-specific localization of these nuclear factors.