Enzymatic detection systems for non-isotopic in situ hybridization using biotinylated cDNA probes.
The feasibility of various non-isotopic enzymatic detection systems was tested for in situ hybridization using biotin-labelled, nick-translated cDNA probes. For this purpose, we isolated and prepared cDNA restriction fragments encoding the proteolytic cysteine proteinase cathepsin L and analysed Kirsten murine sarcoma virus-transformed BALB/3T3 cells, which have been shown to express high amounts of cytoplasmic RNA of this ras oncogene-induced proteinase. When compared on a semiquantitative basis, colorimetric non-isotopic detection of cDNA hybrids with avidin-biotin-peroxidase conjugates visualized by silver intensification of the nickel-diaminobenzidine end-product was superior to that obtained with avidin-biotin-alkaline phosphatase using different substrates for development. When the peroxidase staining technique was applied for RNA detection, it was found that overnight incubation in methanol containing hydrogen peroxide followed by deproteination with HCl was the most effective method for inhibition of endogenous peroxidase activity. For DNA detection, non-specific nucleic staining was completely abolished when heat treatment (100 degrees C) of the cell specimens was performed prior to hybridization.
Trabandt
A
A
University of Alabama at Birmingham, Division of Clinical Immunology and Rheumatology 35294, USA.
Gay
R E
RE
Sikhatme
V P
VP
Gay
S
S
D000469:Alkaline Phosphatase; D000818:Animals; D001360:Avidin; D001426:Bacterial Proteins; D001710:Biotin; D002403:Cathepsins; D003124:Colorimetry; D015342:DNA Probes; D004792:Enzyme Precursors; D011905:Genes, ras; D015151:Immunoblotting; D017403:In Situ Hybridization / Q000379:methods*; D051379:Mice; D008807:Mice, Inbred BALB C; D010544:Peroxidases; D012367:RNA, Viral / Q000096:biosynthesis; D009053:Sarcoma Viruses, Murine / Q000378:metabolism; D019809:Streptavidin; D014407:Tumor Cells, Cultured
D001426:Bacterial Proteins; D015342:DNA Probes; D004792:Enzyme Precursors; D012367:RNA, Viral; D001360:Avidin; D001710:Biotin; D019809:Streptavidin; D010544:Peroxidases; D000469:Alkaline Phosphatase; D002403:Cathepsins; C055808:preprocathepsin L