Using a Multiplex Nucleic Acid in situ Hybridization Technique to Determine HCN4 mRNA Expression in the Adult Rodent Brain

Nonspecific binding of a number of unrelated nucleic acid probes to cells in the crypts of Lieberkuhn was observed in the small intestine of mice with the in situ hybridization technique. Hybridization signal was localized to cells which, by virtue of their histological position, represented Paneth cells. This signal could not be removed by RNAse, DNAse, or proteinase K treatment, and was not removed after high-stringency washing conditions. This report indicates that caution must be exercised in the interpretation of in situ hybridization data when looking for nucleic acid sequences in the gastrointestinal tract.

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Peptide-regulated guanylate cyclase pathways in rat colon: in situ localization of GCA, GCC, and guanylin mRNA

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1993.265.2.g394 ◽

1993 ◽

Vol 265 (2) ◽

pp. G394-G402 ◽

Cited By ~ 11

Author(s):

Z. Li ◽

M. F. Goy

Keyword(s):

In Situ Hybridization ◽

Mrna Expression ◽

Natriuretic Peptide ◽

Proximal Colon ◽

Bacterial Toxin ◽

Surface Epithelium ◽

Rat Colon ◽

Hybridization Technique ◽

In Cells

Guanylate cyclases play a role in both physiological and pathological secretion in the mammalian intestine. Agents that raise guanosine 3',5'-cyclic monophosphate (cGMP) levels, such as atrial natriuretic peptide (ANP), guanylin (an endogenous intestinal peptide), or Escherichia coli heat-stable enterotoxin type a (STa; a bacterial toxin), enhance electrolyte secretion and the accumulation of luminal fluid. Although secretion in all parts of intestine is sensitive to changes in cGMP metabolism, an increasing body of evidence suggests that these responses are particularly important in proximal colon. To date, three peptide-sensitive membrane-bound guanylate cyclases [types A, B, and C (GCA, GCB, and GCC, respectively)] have been cloned from mammalian tissues. GCA responds to ANP, GCB to C-type natriuretic peptide, and GCC to guanylin and STa. Expression of these receptor/cyclase genes has not previously been investigated at the cellular level in the colon. Nucleotide probes specific for GCA, GCB, GCC, and guanylin were generated by polymerase chain reaction. These probes were used to evaluate colonic cyclase and guanylin mRNA expression in the rat. GCB mRNA is not detectable in this tissue either by in situ hybridization or by Northern blot analysis. In contrast, GCA, GCC, and guanylin mRNAs are all conspicuously expressed. With the in situ hybridization technique, GCA mRNA expression is seen in cells in the lamina propria. GCC mRNA expression is seen in epithelial cells throughout colonic crypts, and also, although at a slightly lower level, in cells of the surface epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)

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Localization of Classical Swine Fever Virus from Chronically Infected Pigs by In Situ Hybridization and Immunohistochemistry

Veterinary Pathology ◽

10.1354/vp.40-1-107 ◽

2003 ◽

Vol 40 (1) ◽

pp. 107-113 ◽

Cited By ~ 14

Author(s):

C. Choi ◽

C. Chae

Keyword(s):

In Situ Hybridization ◽

Nucleic Acid ◽

Mononuclear Cells ◽

Classical Swine Fever ◽

Lymphoid Tissues ◽

Hybridization Technique ◽

Tissue Sections ◽

Background Staining ◽

The Central Nervous System

Classical swine fever (CSF) virus (CSFV) nucleic acid and antigen were detected in 15 pigs with naturally occurring chronic CSF by in situ hybridization and immunohistochemistry. The most consistent and prominent microscopic lesions were perivascular mononuclear cell infiltration and gliosis in the central nervous system of pigs with chronic CSF. Positive cells typically exhibited a dark brown (in situ hybridization) or red (immunohistochemistry) reaction product in the cytoplasm without background staining. A positive signal for both in situ hybridization and immunohistochemistry was detected in mononuclear cells and lymphocytes of lymphoid tissues. Viral nucleic acid was detected in some tissue sections in the absence of viral antigen. The in situ hybridization technique developed in this study was useful for the detection of CSFV RNA in tissues taken from chronically infected pigs and may be a valuable technique for studying the pathogenesis of chronic CSFV infection.

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Postoperative differentiation between unilateral adrenal adenoma and bilateral adrenal hyperplasia in primary aldosteronism by mRNA expression of the gene CYP11B2

Acta Endocrinologica ◽

10.1530/eje.0.1510073 ◽

2004 ◽

pp. 73-85 ◽

Cited By ~ 43

Author(s):

U Enberg ◽

C Volpe ◽

A Hoog ◽

A Wedell ◽

LO Farnebo ◽

...

Keyword(s):

In Situ Hybridization ◽

Mrna Expression ◽

Clinical Data ◽

Primary Aldosteronism ◽

Zona Glomerulosa ◽

Hybridization Technique ◽

Steroidogenic Enzyme ◽

Aldosterone Production

OBJECTIVE: Primary aldosteronism (PA) is characterized by hypertension, hypokalemia and suppressed renin-angiotensin system caused by autonomous aldosterone production. The aim of this study was to localize mRNA expression of the genes coding for steroidogenic enzymes in adrenals from a group of patients with PA and relate this to clinical work-up, histopathology and outcome of adrenalectomy. DESIGN: This was a retrospective study of 27 patients subjected to adrenalectomy for PA. METHODS: Clinical data were collected and follow-up of all patients was performed. Paraffin-embedded specimens were analyzed by the in situ hybridization technique, with oligonucleotide probes coding for the steroidogenic enzyme genes. RESULTS: The resected adrenals had the histopathologic diagnosis of adenoma (11), adenoma and/or hyperplasia (15) or hyperplasia (1). CYP11B2 expression (indicating aldosterone production) was found in a dominant adrenal nodule from 22 patients. Fourteen of these had additional CYP11B2 expression in the zona glomerulosa. All 22 patients were cured of PA by adrenalectomy. One of these patients, who had additional high expression of CYP11B2 in the zona glomerulosa, was initially cured, but the condition had recurred at follow-up. Two patients had a mass shown on computed tomography without CYP11B2 but with CYP11B1 and CYP17 expression (indicating cortisol production). Instead their adrenals contained small nodules with CYP11B2 expression. These patients were not cured. CONCLUSIONS: Clinical data, endocrinologic evaluation and histopathology in combination with mRNA in situ hybridization of steroidogenic enzyme genes provide improved opportunities for correct subclassification postoperatively of patients with primary aldosteronism. At present, the in situ hybridization method is of special value for analysis of cases not cured by adrenalectomy.

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In situ hybridization technique to localize rRNA and mRNA in mammalian neurons.

Journal of Histochemistry & Cytochemistry ◽

10.1177/34.1.3941265 ◽

1986 ◽

Vol 34 (1) ◽

pp. 45-50 ◽

Cited By ~ 58

Author(s):

J T McCabe ◽

J I Morrell ◽

R Ivell ◽

H Schmale ◽

D Richter ◽

...

Keyword(s):

In Situ Hybridization ◽

Nucleic Acid ◽

Current Method ◽

Rnase A ◽

Hybridization Technique ◽

Pbr322 Plasmid ◽

Fresh Frozen ◽

Parvocellular Neurons ◽

Plasmid Probe

In situ hybridization provides a method for identifying cells that contain specific nucleic acid sequences. This report outlines an in situ hybridization procedure for mammalian neural tissue. The method maintains morphological quality and produces excellent specificity. Seven tritiated nucleic acid probes were examined: two ribosomal RNA probes, a control pBR322 plasmid probe, two probes encoding portions of the gene for oxytocin, one probe each encoding a portion of vasopressin glycoprotein, and neurophysin. Using cryostat-cut rat brain sections, rRNA probes labeled the cytoplasm of all cells and the nucleoli of larger neurons. The plasmid probe failed to produce a strong signal. Oxytocin and vasopressin probes appropriately labeled the cytoplasm of hypothalamic magnocellular neurons. Vasopressin parvocellular neurons were not identified by the current method, and the shorter length neurophysin probe failed to produce a signal. Methodological variables were examined by counting autoradiographic grains in cells. The longer oxytocin probe produced a stronger signal than the shorter oxytocin and vasopressin probes, and higher probe concentrations resulted in stronger signal. Hybridization could be abolished by tissue pretreatment with RNAse A, and longer exposure time increased signal strength. The outlined fixation steps with fresh-frozen tissue produced a superior signal compared to paraformaldehyde-perfused tissue.

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Histopathological study on the prevalence of trichosporonosis among autopsy subjects using peptide nucleic acid probe In situ hybridization technique

10.26226/morressier.5ac39997d462b8028d89a28a ◽

2018 ◽

Author(s):

Sota Sadamoto

Keyword(s):

In Situ Hybridization ◽

Nucleic Acid ◽

Peptide Nucleic Acid ◽

Histopathological Study ◽

Hybridization Technique ◽

Peptide Nucleic Acid Probe

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Role of hypothalamic ACTH and α-MSH in appetite regulation – an in situ hybridization study of BDNF and MC4-receptor mRNA expression

Experimental and Clinical Endocrinology & Diabetes ◽

10.1055/s-2007-972393 ◽

2007 ◽

Vol 115 (S 1) ◽

Author(s):

K Paulus ◽

C Schulz ◽

H Lehnert

Keyword(s):

In Situ Hybridization ◽

Mrna Expression ◽

Appetite Regulation ◽

Hybridization Study ◽

Receptor Mrna

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Combination of Direct Viable Count and Fluorescent In Situ Hybridization (DVC-FISH) as a Potential Method for Identifying Viable Vibrio parahaemolyticus in Oysters and Mussels

Foods ◽

10.3390/foods10071502 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1502

Author(s):

Jorge García-Hernández ◽

Manuel Hernández ◽

Yolanda Moreno

Keyword(s):

In Situ Hybridization ◽

Vibrio Parahaemolyticus ◽

Fluorescent In Situ Hybridization ◽

Potential Method ◽

Viable Count ◽

Hybridization Technique ◽

Fish Technique ◽

Viable Cells

Vibrio parahaemolyticus is a human food-borne pathogen with the ability to enter the food chain. It is able to acquire a viable, non-cultivable state (VBNC), which is not detected by traditional methods. The combination of the direct viable count method and a fluorescent in situ hybridization technique (DVC-FISH) makes it possible to detect microorganisms that can present VBNC forms in complex samples The optimization of the in vitro DVC-FISH technique for V. parahaemolyticus was carried out. The selected antibiotic was ciprofloxacin at a concentration of 0.75 μg/mL with an incubation time in DVC broth of 5 h. The DVC-FISH technique and the traditional plate culture were applied to detect and quantify the viable cells of the affected pathogen in artificially contaminated food matrices at different temperatures. The results obtained showed that low temperatures produced an important logarithmic decrease of V. parahaemolyticus, while at 22 °C, it proliferated rapidly. The DVC-FISH technique proved to be a useful tool for the detection and quantification of V. parahaemolyticus in the two seafood matrices of oysters and mussels. This is the first study in which this technique has been developed to detect viable cells for this microorganism.

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