Amplification of the in situ hybridization signal by silver postintensification: the biotin-dUTP-streptavidin-peroxidase diaminobenzidine-silver-gold detection system.
Frozen and vibratome sections from the adrenal gland of the rat were hybridized in situ using a biotinylated oligonucleotide probe specific for tyrosine hydroxylase (TH) messenger ribonucleic acid (mRNA). Hybridization was detected using the streptavidin-peroxidase-diaminobenzidine (DAB) system in combination with silver-gold postintensification. The signal appeared as a black coloration and was localized to the cytoplasm of catecholamine-synthesizing chromaffin cells in the adrenal medulla. This coloration was due to the deposition of the silver-gold intensified DAB chromogen onto the probe hybridized to mRNA in carrier organelles. Compared with the conventional peroxidase-DAB labelling, the silver-gold amplified version was more sensitive in detecting TH mRNA. Using this modification, we were able to adapt the procedure to electron microscopy, thereby further localizing the hybridized signal to ribosomes. Because this hybridization detection system produces grains, not just color, this method has the potential for measurement of changes in mRNA levels at the ultrastructural level.
Department of Anatomy, University Medical School, Pécs, Hungary.
Z
Z
Liposits
S L
SL
Petersen
W K
WK
Paull
D015100:3,3'-Diaminobenzidine; D000313:Adrenal Medulla / Q000737:chemistry* / Q000201:enzymology / Q000648:ultrastructure; D000818:Animals; D001426:Bacterial Proteins; D001483:Base Sequence; D002837:Chromaffin Granules / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D006046:Gold; D006651:Histocytochemistry; D008297:Male; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009195:Peroxidase; D015347:RNA Probes; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D051381:Rats; D011919:Rats, Inbred Strains; D012270:Ribosomes / Q000737:chemistry; D012834:Silver; D019809:Streptavidin; D014446:Tyrosine 3-Monooxygenase / Q000032:analysis / Q000235:genetics*
D001426:Bacterial Proteins; D015347:RNA Probes; D012333:RNA, Messenger; D015100:3,3'-Diaminobenzidine; D012834:Silver; D006046:Gold; D019809:Streptavidin; D009195:Peroxidase; D014446:Tyrosine 3-Monooxygenase