Sensitive mRNA detection using unfixed tissue: combined radioactive and non-radioactive in situ hybridization histochemistry

Dagerlind, Å.; Friberg, Kristina; Bean, Andrew J.; Hökfelt, Tomas

doi:10.1007/bf00716936

Cited by 562 publications

(290 citation statements)

References 40 publications

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“…Hybridization was carried out according to published procedures (Dagerlind et al, 1992) on a minimum of 5 slides/probe from each of the experimental and control groupings. Briefly, the sections were hybridized at 43 °C for 14-18 hours in a buffer containing 50% formamide (Sigma Aldrich, Oakville, ON, Canada), 4X SSC (1X SSC -0.15 M NaCl, 0.015 sodium citrate), 1X Denhart's solution (0.02% bovine serum albumin and 0.02% Ficoll), 1% sarcosyl (N-laurylsarcosine), 0.02 M phosphate buffer (pH 7.0), 10% dextran sulphate, 500 μg/ml heatdenatured sheared salmon sperm DNA, 200 mM dithiothreitol and 10 7 cpm/ml of probe.…”

Section: In Situ Hybridizationmentioning

confidence: 99%

Neurotrophin-3 significantly reduces sodium channel expression linked to neuropathic pain states

Wilson-Gerwing

Stucky

McComb

et al. 2008

Experimental Neurology

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Neuropathic pain resulting from chronic constriction injury (CCI) is critically linked to sensitization of peripheral nociceptors. Voltage gated sodium channels are major contributors to this state and their expression can be upregulated by nerve growth factor (NGF). We have previously demonstrated that neurotrophin-3 (NT-3) acts antagonistically to NGF in modulation of aspects of CCI-induced changes in trkA-associated nociceptor phenotype and thermal hyperalgesia. Thus, we hypothesized that exposure of neurons to increased levels of NT-3 would reduce expression of Na v 1.8 and Na v 1.9 in DRG neurons subject to CCI. In adult male rats, Na v 1.8 and Na v 1.9 mRNAs are expressed at high levels in predominantly small to medium size neurons. One week following CCI, there is reduced incidence of neurons expressing detectable Na v 1.8 and Na v 1.9 mRNA, but without a significant decline in mean level of neuronal expression, and similar findings observed immunohistochemically. There is also increased accumulation/redistribution of channel protein in the nerve most apparent proximal to the first constriction site. Intrathecal infusion of NT-3 significantly attenuates neuronal expression of Na v 1.8 and Na v 1.9 mRNA contralateral and most notably, ipsilateral to CCI, with a similar impact on relative protein expression at the level of the neuron and constricted nerve. We also observe reduced expression of the common neurotrophin receptor p75 in response to CCI that is not reversed by NT-3 in small to medium sized neurons and may confer an enhanced ability of NT-3 to signal via trkA, as has been previously shown in other cell types. These findings are consistent with an analgesic role for NT-3.

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Section: In Situ Hybridizationmentioning

confidence: 99%

Neurotrophin-3 significantly reduces sodium channel expression linked to neuropathic pain states

Wilson-Gerwing

Stucky

McComb

et al. 2008

Experimental Neurology

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“…In situ hybridization was performed as previously described (Dagerlind et al, 1992). Brie¯y, three synthetic oligonucleotides were generated: 5'-CGATCCTTGAAGTAGGA-GCGGCTGAGGCTGTTCCATATGA-3' (Oligo 1); 5'-GA-GCTGTCCAGTTTGGTGCCTGTGATGAAGCTGAAGA-GGGACT-3' (oligo 2) and 5'-CTGCTTCTTCATCTG-CACTTGCGACTGTGCCGTGAGTTGCAG -3' (Oligo 3).…”

Section: Isolation Of Rna and Northern Blot Analysismentioning

confidence: 99%

Characterization of the mouse Men1 gene and its expression during development

et al. 1998

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The gene responsible for multiple endocrine neoplasia type 1 (MEN1), a heritable predisposition to endocrine tumours in man, has recently been identi®ed. Here we have characterized the murine homologue with regard to cDNA sequence, genomic structure, expression pattern and chromosomal localisation. The murine Men1 gene spans approximately 6.7 kb of genomic DNA and is comprised of 10 exons with similar genomic structure to the human locus. It was mapped to the pericentromeric region of mouse chromosome 19, which is conserved with the human 11q13 band where MEN1 is located. The predicted protein is 611 amino acids in length and overall is 97% homologous to the human orthologue. The 45 reported MEN1 mutations which alter or delete a single amino acid in human all occur at conserved residues, thereby supporting their functional signi®cance. Two transcripts of approximately 3.2 and 2.8 kb were detected in both embryonal and adult murine tissues, resulting from alternative splicing of intron 1. By RNA in situ hybridization and Northern analysis the spatiotemporal expression pattern of Men1 was determined during mouse development. Men1 gene activity was detected already at gestational day 7. At embryonic day 14 expression was generally high throughout the embryo, while at day 17 the thymus, skeletal muscle, and CNS showed the strongest signal. In selected tissues from postnatal mouse Men1 was detected in all tissues analysed and was expressed at high levels in cerebral cortex, hippocampus, testis, and thymus. In brain the menin protein was detected mainly in nerve cell nuclei, whereas in testis it appeared perinuclear in spermatogonia. These results show that Men1 expression is not con®ned to organs a ected in MEN1, suggesting that Men1 has a signi®cant function in many di erent cell types including the CNS and testis.

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“…This procedure allowed for parallel in situ hybridization of wild-type and knockout sections under absolutely identical conditions assuring meaningful quantification and comparison of hybridization signals. All sections were processed for in situ hybridization according to a modified version of the procedure described by Dagerlind et al (1992). Specific riboprobes were generated by PCR from respective cDNA clones, which were spotted on the MPIP 17k array.…”

Section: Quantitative In Situ Hybridizationmentioning

confidence: 99%

“…After 20 mins of DNase I (Roche, Penzberg, Germany) treatment, the probes were purified by the RNeasy Clean up protocol (Qiagen, Hilden, Germany) and measured in a scintillation counter. For hybridization, sections were pretreated and prehybridized as described previously (Dagerlind et al, 1992). Subsequently, they were hybridized overnight with a probe concentration of 7 Â 10 6 c.p.m./mL at 571C and washed at 651C in 0.1Â SSC and 0.1 mmol/L dithiothreitol.…”

Section: Quantitative In Situ Hybridizationmentioning

confidence: 99%

Expression Profiling Identifies the CRH/CRH-R1 System as a Modulator of Neurovascular Gene Activity

Deussing

Kühne

Pütz

et al. 2007

J Cereb Blood Flow Metab

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Corticotropin-releasing hormone receptor type 1 (CRH-R1)-deficient mice display reduced anxietylike behavior, a chronic corticosterone deficit, and an impaired neuroendocrine stress response caused by disruption of the hypothalamic-pituitary-adrenocortical (HPA) axis. The molecular substrates and pathways of CRH/CRH-R1-dependent signaling mechanisms underlying the behavioral phenotype as well as the consequences of lifelong glucocorticoid deficit remain largely obscure. To dissect involved neuronal circuitries, we performed comparative expression profiling of brains of CRH-R1 mutant and wild-type mice using our custom made MPIP (Max Planck Institute of Psychiatry) 17k cDNA microarray. Microarray analysis yielded 107 genes showing altered expression levels when comparing CRH-R1 knockout mice with wild-type littermates. A significant proportion of differentially expressed genes was related to control of HPA and hypothalamicpituitary-thyroid (HPT) axes reflecting not only the disturbance of the HPA axis in CRH-R1 mutant mice but also the interplay of both neuroendocrine systems. The spatial analysis of regulated genes revealed a prevalence for genes expressed in the cerebral microvasculature. This phenotype was confirmed by the successful cross-validation of regulated genes in CRH overexpressing mice. Analysis of the cerebral vasculature of CRH-R1 mutant and CRH overexpressing mice revealed alterations of functional rather than structural properties. A direct role of the CRH/CRH-R1 system was supported by demonstrating Crhr1 expression in the adult murine cerebral vasculature. In conclusion, these data suggest a novel, previously unknown role of the CRH/CRH-R1 system in modulating neurovascular gene expression and function.

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Sensitive mRNA detection using unfixed tissue: combined radioactive and non-radioactive in situ hybridization histochemistry

Cited by 562 publications

References 40 publications

Neurotrophin-3 significantly reduces sodium channel expression linked to neuropathic pain states

Neurotrophin-3 significantly reduces sodium channel expression linked to neuropathic pain states

Characterization of the mouse Men1 gene and its expression during development

Expression Profiling Identifies the CRH/CRH-R1 System as a Modulator of Neurovascular Gene Activity

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