Failure of heparin to inhibit the expression of the thrombin receptor following endothelial injury of the rabbit carotid artery.

The effect of heparin on thrombin receptor expression was evaluated in an experimental model of myointimal smooth muscle cell proliferation in rabbits. Myointimal hyperplasia was induced by an air-drying injury of the carotid artery and thrombin receptor expression following endothelial injury was measured by in situ hybridisation and immunohistochemistry. In healthy arteries, thrombin receptor mRNA and protein were detected in the endothelial cells only.

Histochemical detection of the messenger RNAs coding for calcitonin and calcitonin gene-related peptide in medullary thyroid carcinomas with radioactive and biotinylated oligonucleotide probes.

The present study has been undertaken to investigate the efficiency of biotinylated synthetic oligonucleotide probes in detection by in situ hybridization of the mRNAs coding for calcitonin (CT) or calcitonin gene-related peptide (CGRP) in human medullary thyroid carcinomas (MTCs). Tissue sections fixed with formaldehyde were hybridized with 45-base long oligonucleotides, specific for CT or CGRP mRNA. Recombinant DNA probe or synthetic oligonucleotides radioactively labelled with 32P or 35S were used as controls to detect by autoradiography the corresponding mRNAs in the tumour cells.

Topography and ontogeny of the neurons expressing vasopressin, oxytocin, and somatostatin genes in the rat brain: an analysis using radioactive and biotinylated oligonucleotides.

1. The use of radioactive and biotinylated oligonucleotide probes has been optimized to detect and analyze by in situ hybridization, neurons expressing neuropeptide genes (vasopressin, oxytocin, somatostatin). 2.

Presence of neuropeptide messenger RNAs in neuronal processes.

The messenger RNAs coding for vasopressin, oxytocin, luteinizing hormone releasing-hormone and somatostatin have been detected in tissue sections of the rat brain, especially in the hypothalamus with radioactive and biotinylated oligonucleotide probes. The results demonstrate that neuropeptide mRNAs are present in the cytoplasm of cell bodies, in processes and in punctate structures in the vicinity of the cell bodies. These results demonstrate that neuropeptide mRNAs can be transported outside the cell body most probably in proximal dendrites but also in some of their branching, and possibly at synaptic contacts.

Dopamine receptor gene expression by enkephalin neurons in rat forebrain.

In situ hybridization experiments were performed with brain sections from normal, control and haloperidol-treated rats to identify and map the cells expressing the D2 dopamine receptor gene. D2 receptor mRNA was detected with radioactive or biotinylated oligonucleotide probes. D2 receptor mRNA was present in glandular cells of the pituitary intermediate lobe and in neurons of the substantia nigra, ventral tegmental area, and forebrain, especially in caudate putamen, nucleus accumbens, olfactory tubercle, and piriform cortex.

Vasopressin gene expression in the normal and Brattleboro rat: a histological analysis in semi-thin sections with biotinylated oligonucleotide probes.

We analyzed expression of the vasopressin (AVP) gene in semi-thin sections in normal and Brattleboro rats by using in situ hybridization and immunohistochemistry. AVP mRNA was detected as follows: vibratome sections of rat hypothalamus were hybridized with a biotinylated oligonucleotide probe, embedded in Araldite, and cut into semi-thin sections which were reacted with streptavidin-alkaline phosphatase and the appropriate substrate. Adjacent serial sections were treated by immunohistochemistry to detect AVP or oxytocin immunoreactivity.

Ultrastructural detection of the vasopressin messenger RNA in the normal and Brattleboro rat.

The messenger RNA coding for vasopressin has been detected at the ultrastructural level in normal and Brattleboro rat neurons, by using an oligonucleotide rat AVP prove labelled with 35SdATP. Vibratome sections of rat hypothalamus fixed with a mixture of 4% formaldehyde and 0.1% glutaraldehyde were hybridized with the probe, osmicated, included in Araldite and cut in semi-thin and thin sections that were coated with emulsion.

Analysis of vasopressin gene expression by in situ hybridization and immunohistochemistry in semi-thin sections.

We have designed a procedure to investigate vasopressin (AVP) gene expression on plastic-embedded tissue by using in situ hybridization to detect AVP mRNA and immunohistochemistry to detect AVP. Rat brain was fixed and vibratome slices were incubated with a 45-base synthetic oligonucleotide complementary to AVP mRNA labeled with 35S, embedded in Araldite, and cut into semi-thin serial sections that were either processed for autoradiography or treated with an AVP antiserum. The results show that AVP mRNA is detectable in magnocellular neurons of the supraoptic and paraventricular nuclei in both vibratome and semi-thin sections.

Histological detection of messenger RNAs with biotinylated synthetic oligonucleotide probes.

We achieved histological detection of the messenger RNAs coding for vasopressin, calcitonin, or calcitonin gene-related peptide by using biotinylated synthetic oligonucleotides, and defined the technical parameters enabling optimal detection of these mRNAs. Oligonucleotides labeled by fixation of one biotin at their 5' end or by addition of a biotin-11-dUTP tail at their 3' end can be used to detect mRNAs, although the latter are more sensitive. Streptavidin-alkaline phosphatase revealed with nitroblue tetrazolium-bromo-chloro-indolyl phosphate as substrate makes possible detection of the biotinylated oligonucleotides.

In situ hybridization histochemistry for the analysis of gene expression in the endocrine and central nervous system tissues: a 3-year experience.

We report our experience in development of the in situ hybridization (ISH) procedure to detect messenger RNAs (mRNAs) coding for various molecules involved in endocrine glands and central nervous system activity, including mRNAs coding for endorphin precursors [preproenkephalin A (PPA), pro-opiocortin (POMC)], vasopressin, and transferrin. Various conditions of fixation and handling of the tissues were tested to establish optimal parameters for mRNA detection. Double-stranded DNA probes labeled by nick translation, synthetic oligonucleotides labeled at their 5' end, as well as single-stranded RNA probes were used, after incorporation of 32P- or 35S-labeled nucleotides.