Localization of nuclear RNA by pre- and post-embedding in situ hybridization using different gold probes.
Pre-embedding and post-embedding in situ hybridization techniques were compared for the localization of RNAs in the nucleus. 28S rRNA and transcripts of the epidermal growth factor receptor (EGF-receptor) were localized with both hybridization methods. Pre-embedding hybridizations were performed on cells permeabilized with Triton X-100, whereas post-embedding hybridizations were carried out on Lowicryl K4M sections. From these studies it was concluded that, for labelling of 28S rRNA, the post-embedding in situ hybridization is preferred, whereas EGF-receptor transcripts were successfully detected only after pre-embedding in situ hybridization. Furthermore, the detection of the hybrids with ultra-small gold particles was compared to the detection with 6 nm gold particles in both pre- and post-embedding in situ hybridization studies. From our results it is concluded that the use of ultra-small gold particles results in higher label efficiency. Therefore, ultra-small gold particles are preferable to 6 nm gold particles for the detection of hybrids in high-resolution in situ hybridization experiments.
Sibon
O C
OC
Department of Molecular Cell Biology, University of Utrecht, The Netherlands.
Cremers
F F
FF
Humbel
B M
BM
Boonstra
J
J
Verkleij
A J
AJ
D002467:Cell Nucleus / Q000737:chemistry; D015342:DNA Probes; D066246:ErbB Receptors / Q000235:genetics; D006046:Gold; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D010316:Particle Size; D012313:RNA / Q000032:analysis*; D015347:RNA Probes; D012339:RNA, Ribosomal, 28S / Q000032:analysis; D016610:Tissue Embedding; D014407:Tumor Cells, Cultured
D015342:DNA Probes; D015347:RNA Probes; D012339:RNA, Ribosomal, 28S; D012313:RNA; D006046:Gold; D066246:ErbB Receptors