Ultrastructural localization of epidermal growth factor (EGF)-receptor transcripts in the cell nucleus using pre-embedding in situ hybridization in combination with ultra-small gold probes and silver enhancement.
A high-resolution in situ hybridization method is described for localizing epidermal growth factor (EGF)-receptor transcripts in nuclei of A431 epidermoid carcinoma cells. The method is based upon the use of ultra-small gold particles in combination with silver enhancement. The RNA of the EGF-receptor was detected mainly around the nucleoli. After removal of the DNA using nucleases and high salt extraction, the RNA of the EGF-receptor appears to be associated with the nuclear matrix. The RNA of the EGF-receptor was observed in close contact with the SC-35 splicing protein, but no exact colocalization was observed. These results demonstrate that high resolution pre-embedding in situ hybridization in combination with immunocytochemistry, both using ultra-small gold as a detection method, provides a powerful tool to unravel the organization of nuclear processes.
Sibon
O C
OC
Department of Molecular Cell Biology, University of Utrecht, The Netherlands.
Humbel
B M
BM
De Graaf
A
A
Verkleij
A J
AJ
Cremers
F F
FF
D016475:3T3 Cells; D000818:Animals; D002294:Carcinoma, Squamous Cell; D002460:Cell Line; D002466:Cell Nucleolus / Q000378:metabolism / Q000648:ultrastructure; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure; D066246:ErbB Receptors / Q000032:analysis / Q000096:biosynthesis*; D006046:Gold; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods; D051379:Mice; D008854:Microscopy, Electron / Q000379:methods; D015530:Nuclear Matrix / Q000378:metabolism / Q000648:ultrastructure; D015347:RNA Probes; D012834:Silver; D013194:Staining and Labeling; D014407:Tumor Cells, Cultured
D015347:RNA Probes; D012834:Silver; D006046:Gold; D066246:ErbB Receptors