A comparative study of digoxigenin, 2,4-dinitrophenyl, and alkaline phosphatase as deoxyoligonucleotide labels in non-radioisotopic in situ hybridisation.

Abstract

Aim-To determine the optimum form of labelling and the most efficient reporter molecule for non-radioisotopic in situ hybridisation (ISH). Methods-Nine deoxyoligonucleotides complementary to histone mRNA were synthesised and labelled either enzymatically or during solid-phase synthesis with the reporter molecules digoxigenin, 2,4-dinitrophenyl (DNP), or alkaline phosphatase. Pooled deoxyoligonucleotide cocktails were then used in nonradioisotopic ISH detection of histone mRNA in human tonsil. Hybrid detection was by nitroblue tetrazoleum/5-bromo-4-chloro-3-indolyl phosphate colorimetric development. Results-The use of a spacer in 3' enzymatic labelling and when labelling with alkaline phosphatase significantly increased ISH signal. The 3' and 5' labelling of oligonucleotides with triple DNP groups during solid-phase synthesis produced the strongest signal as determined by the highest cell signal intensity and shortest development time. Conclusions-3' and 5' solid-phase labelling with triple DNP groups produced the best labelling for non-isotopic ISH using deoxyoligonucleotide cocktails. (T Clin Pathol 1997;50:686-690)

Differentiated human podocytes endogenously express an inhibitory isoform of vascular endothelial growth factor (VEGF165b) mRNA and protein

“…difference between full-length mRNAs is only 18 bases. A single oligonucleotide (for instance, across exon 7-8b splice site) will not produce enough visible signal isotopically or after optimization of hapten-labeling and detection as we previously described (15). Furthermore, an exon 8b-containing sequence is present in both stimulatory and inhibitory VEGF families, as part of the 3ЈUTR and as coding sequence, respectively.…”

Heterogeneous vascular endothelial growth factor (VEGF) isoform mRNA and receptor mRNA expression in human glomeruli, and the identification of VEGF148 mRNA, a novel truncated splice variant

“…Although it would be preferable to confirm this heterogeneity in the relative expression of VEGF isoforms and receptors between different glomeruli using a technique such as in situ hybridization, this has not been possible. Despite considerable experience in developing new strategies with non-isotopic in situ hybridization [18][19][20][21][22][23][24], we have not been able to develop a probe cocktail that will distinguish between the VEGF isoform mRNAs. This is primarily because of the striking similarity between isoforms, with the only sequence specific for each being the spliced exon-exon boundaries.…”

Vascular endothelial growth factor mRNA expression in minimal change, membranous, and diabetic nephropathy demonstrated by non-isotopic in situ hybridisation

“…Unfortunately standard non-isotopic ISH is not sensitive enough to work in these circumstances, despite the newer hapten labels. 9 To add to the complexity, there are at least three receptors for VEGF. One of these, soluble-Flt, is a form which inactivates VEGF when bound.…”

“…9 10 RNase-free reagents and glassware (diethylpyrocarbonate (DEPC) treated; Sigma; D5758) were used throughout. Sections (4 µm) were cut onto silane coated slides.…”