Development of a Sensitive and Specific in Situ Hybridization Technique for the Cellular Localization of Antisense Oligodeoxynucleotide Drugs in Tissue Sections

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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Whole-mount in situ hybridization: minimizing the folding problem of thin-sheet tissue-like crayfish haematopoietic tissue

Crustaceana ◽

10.1163/15685403-00003745 ◽

2018 ◽

Vol 91 (1) ◽

pp. 1-15

Author(s):

Aleksandra Zečić ◽

Chadanat Noonin

Keyword(s):

In Situ Hybridization ◽

Histological Study ◽

Thin Sheet ◽

Hybridization Technique ◽

Tissue Sections ◽

Specific Transcript ◽

Optimized Protocol ◽

Whole Mount ◽

Haematopoietic Tissue

Crayfish haematopoietic tissue (HPT) has a thin-sheet-like structure with a thickness of 100-160 μm and a width of approximately 1-2 cm. This structure makes HPT extremely easy to fold after removal from the animal. Therefore, it is difficult to handle the tissue without folding when processing for sectioning and histological study. The degree of tissue folding reflects the size of the tissue sections obtained, how complicated it is to interpret the location of each tissue section, and the accuracy of the interpretation of the location of a specific transcript. To facilitate the interpretation of a specific transcript location in the HPT, we optimized a whole-mount in situ hybridization technique to minimize tissue folding. This optimized protocol effectively reduced the tissue folding. Therefore, the location of a specific transcript in the HPT was easily and accurately defined. This protocol will be useful for whole-mount staining of other tissues with similar structure.

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Cellular localization of apolipoprotein D and lecithin:cholesterol acyltransferase mRNA in rhesus monkey tissues by in situ hybridization.

The Journal of Lipid Research ◽

10.1016/s0022-2275(20)42739-7 ◽

1990 ◽

Vol 31 (6) ◽

pp. 995-1004

Author(s):

KM Smith ◽

RM Lawn ◽

JN Wilcox

Keyword(s):

In Situ Hybridization ◽

Rhesus Monkey ◽

Cellular Localization ◽

Lecithin:Cholesterol Acyltransferase ◽

Apolipoprotein D

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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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Cellular Localization of Inducible Nitric Oxide Synthase in Experimental Endotoxic Shock in the Rat

Clinical Science ◽

10.1042/cs0870179 ◽

1994 ◽

Vol 87 (2) ◽

pp. 179-186 ◽

Cited By ~ 67

Author(s):

H. Terence Cook ◽

Alison J. Bune ◽

Albertine S. Jansen ◽

G. Michael Taylor ◽

Rashpal K. Loi ◽

...

Keyword(s):

Nitric Oxide ◽

In Situ Hybridization ◽

Cellular Localization ◽

Endotoxic Shock ◽

No Synthase ◽

Similar Distribution ◽

Mouse Macrophage ◽

Inducible No Synthase ◽

Monocytes And Macrophages

1. Endotoxin induces a shock-like syndrome with increased nitric oxide synthesis. To clarify the cellular source of NO in endotoxic shock we used immunohistochemistry and in situ hybridization to localize inducible NO synthase in rats given lipopolysaccharide or Corynebacterium parvum and lipopolysaccharide. Immunohistochemistry was carried out with an antibody raised against a synthetic peptide of mouse macrophage NO synthase. In situ hybridization was performed with 35S-labelled oligonucleotide probes corresponding to cDNA sequences common to mouse macrophage inducible NO synthase and rat vascular smooth inducible NO synthase. Monocytes and macrophages were identified by immunohistochemistry with the mouse monoclonal antibody ED1. 2. After lipopolysaccharide alone, the major site of NO synthase induction was monocytes and macrophages in multiple organs, principally liver and spleen. Bronchial, bile duct, intestinal and bladder epithelium and some hepatocytes also expressed inducible NO synthase. Expression peaked at 5 h and had returned to normal by 12 h except in spleen. 3. After priming with C. parvum, lipopolysaccharide led to a similar distribution of inducible NO synthase as lipopolysaccharide alone, but in addition there was more prominent hepatocyte staining, staining in macrophage granulomas in the liver and inducible NO synthase was present in some endothelial cells in the aorta. 4. These findings provide a direct demonstration of the cellular localization of inducible NO synthase after lipopolysaccharide.

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Microbial ecology of sulfate-reducing bacteria in wastewater biofilms analyzed by microelectrodes and fish (fluorescent in situ hybridization) technique

Water Science & Technology ◽

10.2166/wst.1999.0323 ◽

1999 ◽

Vol 39 (7) ◽

pp. 41-47 ◽

Cited By ~ 3

Author(s):

Satoshi Okabe ◽

Hisashi Satoh ◽

Tsukasa Itoh ◽

Yoshimasa Watanabe

Keyword(s):

In Situ Hybridization ◽

Sulfate Reduction ◽

Sulfate Reducing Bacteria ◽

Hybridization Technique ◽

Concentration Profiles ◽

Reduction Zone ◽

Sulfate Reducing ◽

Reducing Bacteria ◽

Culture Dependent

The vertical distribution of sulfate-reducing bacteria (SRB) in microaerophilic wastewater biofilms grown on fully submerged rotating disk reactors (RDR) was determined by the conventional culture-dependent MPN method and in situ hybridization of fluorescently-labelled 16S rRNA-targeted oligonucleotide probes for SRB in parallel. Chemical concentration profiles within the biofilm were also measured using microelectrodes for O2, S2-, NO3- and pH. In situ hybridization revealed that the SRB probe-stained cells were distributed throughout the biofilm even in the oxic surface zone in all states from single scattered cells to clustered cells. The higher fluorescence intensity and abundance of SRB probe-stained cells were found in the middle part of the biofilm. This result corresponded well with O2 and H2S concentration profiles measured by microelectrodes, showing sulfate reduction was restricted to a narrow anaerobic zone located about 500 μm below the biofilm surface. Results of the MPN and potential sulfate reducing activity (culture-dependent approaches) indicated a similar distribution of cultivable SRB in the biofilm. The majority of the general SRB probe-stained cells were hybridized with SRB 660 probe, suggesting that one important member of the SRB in the wastewater biofilm could be the genus Desulfobulbus. An addition of nitrate forced the sulfate reduction zone deeper in the biofilm and reduced the specific sulfate reduction rate as well. The sulfate reduction zone was consequently separated from O2 and NO3- respiration zones. Anaerobic H2S oxidation with NO3- was also induced by addition of nitrate to the medium.

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