Navigation Links
Detection of mRNAs on Cryosections of the,,,Cardiovascular System Using DIG-Labeled RNA Probes

The following protocol was optimized from a protocol using 35S-labeled RNA probes [1, 2]. In research studies, it enables detection of the expression of rare mRNAs in the cardiovascular system (e.g., of the proinflammatory cytokine granulocyte macrophage colony stimulating factor [GM-CSF] in normal human coronary arteries, and of interleukin 6 [IL6] and glycoprotein gp130 in failing human hearts [3]). The protocol can be combined with immunohistochemistry [4, 5], and can also be used for paraffin- and methacrylate-embedded sections.

Materials and Methods

Preparation of DIG-labeled RNA probes

The desired cDNA was amplified by reverse transcription polymerase chain reaction (RT-PCR), and the cDNA was cloned into an in vitro transcription vector. DIG-labeled RNA probes were transcribed in vitro from the plasmid according to the instructions of the DIG RNA Labeling Kit. Best results will be obtained with digoxigenin-labeled RNA probes of < 600 bp. For larger probes, adjusting the proteinase K digest is preferred to probe denaturation. The amount of DIG-labeling was determined by dot blot. It is important to determine the exact concentration of the RNA probes to avoid background after hybridization.

Tissue preparation

Tissue was frozen as soon as possible after excision to prevent degradation of mRNA. The tissue was cut to the appropriate size, and as much of the fatty tissue as possible was removed. The tissue was then immersed in a cryoprotective medium and frozen on cork disks in nitrogen- cooled 2-methylbutan. The tissue was stored at 80 C or in liquid nitrogen.

Preparation of cryosections
The tissue samples were prewarmed to 22 C and tissue sections were prepared (4 m12 m, thicker sections may be preferred for confocal microscopy). The sections (24) were placed on silanecoated slides and immediately used or stored at 80 C (up to several months).

Prehybridization procedure

The slides were dried for 1 hour at room temperature or in an oven for 10 minutes at 50 C. The sections were treated for 10 minutes with chloroform, if the tissue was rich in lipids. The sections were fixed for 10 minutes with phosphate-buffered 4% paraformaldehyde and rinsed 3 times in 5x TE (50 mM Tris-HCl pH 8.0, 5 mM EDTA). The sections were treated with proteinase K for 10 minutes at room temperature if necessary. Whether proteinase K treatment is required, and which concentration of proteinase K is used strongly depends on the kind of tissue and the fixation. For blood vessels and myocardial tissue we used:
  • Cryosections: up to 2 g/ml
  • Paraffin-embedded sections: up to 20 g/ml
  • Methacrylate-embedded sections: up to 50 g/ml
Sections were rinsed in Tris-Glycine (100 mM Tris-HCl pH7.0, 100 mM glycine), post-fixed for 10 minutes in 4% phosphate-buffered paraformaldehyde, and rinsed in TBS (50 mM Tris-HCl pH 7.5, 150 mM NaCl) 3 times for 5 minutes. The sections were then rinsed in distilled water once (5 minutes), dehydrated in increasing concentrations of ethanol, and dried in a dust-free area. After the prehybridization procedure, the sections may be stored for a few days in a refrigerator. However, optimal results will be achieved by immediately continuing with in situ hybridization.

Hybridization procedure

Homologous probes were hybridized at 5052 C. For heterologous probes, we recommend lower temperatures. The hybridization solution (50% formamide, 2x SSPE buffer, 10mM DTT, 1mg/ml herring sperm DNA, 500 g/ml yeast tRNA, 1 mg/ml BSA) was denatured for 10 minutes at 80 C. The sections were preincubated in a humidified chamber for 2 hours in hybridization solution. The prehybridization solution was removed, and hybridization solution was added (concentration of the DIG-labeled RNA probe: 0.3 1 g/ml). The tightly sealed chambers were incubated overnight in a shaking water bath at 50 C.

Posthybridization procedure

The hybridization solution was removed by thoroughly rinsing the slides in 4x SSC. The slides were washed twice in 2x SSC for 15 minutes at 50 C and twice in 1x SSC for 15 minutes at 50 C. To remove nonspecifically bound DIG-labeled RNA probes, the slides were incubated with RNase A (10 g/ml NTE: 500 mM NaCl, 10mM Tris-HCl pH 8.0, 1mM EDTA) for 10 minutes at 37 C. The slides were then washed twice in 0.1x SSC for 10 minutes at 50 C.

Detection procedure

For detection, the DIG Nucleic Acid Detection Kit was used. The sections were washed for 5 minutes in buffer 1. Nonspecific background was blocked by incubating the sections for 1 hour in buffer 2 (buffer 1 with 0.5% Blocking Reagent).

  • Alkaline phosphatase conjugated antibodies

The sections were incubated with anti-DIG antibody conjugated with alkaline phosphatase (Fab-fragments) (dilute 1:5001:1,000 in buffer 2) for 1 hour at room temperature, then rinsed thoroughly in buffer 1 containing 0.05% Tween, washed twice for 15 minutes, and incubated for another 15 minutes in buffer 3. The slides were then incubated with an appropriate amount of staining solution in a humidified chamber for 30 minutes to 24 hours (staining solution: 335 g NBT [stock solution: 75 mg/ml in 70 % dimethylformamide], 174 g BCIP [stock solution: 50 mg/ml in 100% dimethylformamide], and 240 g Levamisole per ml buffer 3). The staining solution was removed by rinsing in 5x TE, the staining procedure was stopped by incubating the slides for 15 minutes in 5x TE. The sections were briefly rinsed in distilled water, then counterstained with methylene green. The sections were mounted with Kaisers glycerin-gelatin. If immunofluorescence protocols are used in combination with in situ hybridization, we recommend development with the ELF substrate (ELF Kit, Molecular Probes).

  • Fluorochrome-conjugated antibodies

When the mRNA of interest is abundantly expressed, anti-DIG antibodies conjugated with FITC or other fluorochromes can be used. The slides were incubated for 2 hours with FITC-conjugated anti-DIG antibody (1:201:200 in buffer 1), then washed twice for 5 minutes with buffer 2 (Buffer 1 containing 0.05% Tween). The sections were counterstained with Hoechst Dye 33342 and mounted with fluorescence mounting medium (DAKO).

Immunohistochemistry

Usually immunohistochemistry was performed immediately after in situ hybridization. However, with both detection procedures excellent results were also obtained for several weeks (up to months) after in situ hybridization. All washing steps were performed on a shaking platform.

  • The peroxidase staining procedure was performed according to the manufacturers recommendations (Vectastain Elite Kit, Vector Laboratories).
  • Immunofluorescence: The slides were incubated with PBS/12% BSA for 12 hours in a humidified chamber, then the blocking solution was removed. The slides were incubated with an appropriate dilution of the respective primary antibody (in PBS /12% BSA) for 14 hours at room temperature or overnight at 4 C in a humidified chamber. The excess antibody was removed by washing three-times for 5 minutes in PBS that contained 0.05% Tween. The slides were then incubated with an appropriate dilution (1:5001:1000 in PBS /12% BSA) of the secondary antibody (for immunoconfocal detection, we recommend Cy-conjugated antibodies such as provided by Chemicon), then washed three times for 5 minutes in PBS, counterstained with Hoechst Dye 33342, and mounted with fluorescence mounting medium (DAKO).
Results and Discussion

Combined in situ hybridization and immunohistochemical staining has been used to identify vascular cells expressing GM-CSF and type VIII collagen in human coronary arteries. This non-radioactive procedure combined
  • in situ hybridization with DIG-labeled cRNA probes (GM-CSF, type VIII collagen) and
  • immunohistochemical characterization of vascular cells using cell type-specific antibodies (smooth muscle cells: anti smooth muscle actin, Enzo; endothelial cells: von Willebrand factor, Sigma; macrophages: CD68, DAKO) and a peroxidase staining procedure (Vectastain Elite Kit, Vector Laboratories). Approximately 70 % of the antibodies tested worked in this protocol.
The method enabled us to identify the intimal and medial smooth muscle cells as the major cell type expressing granulocyte macrophage colony stimulating factor (GMCSF) in the development of atherosclerotic plaques, particularly in advanced lesions [4]. Other GM-CSF-expressing cell types found in advanced lesions are endothelial cells and macrophages. In early lesions, GM-CSF mRNA (in situ hybridization, purple stain) was expressed mainly by medial smooth muscle cells, some smooth muscle cells of the tunica intima (Figure 1A), about 50 % of the endothelial cells (Figure 1B), and only a few macrophages located in the tunica adventitia (Figure 1C). In all stages of plaque development, GM-CSF was coexpressed with type VIII collagen [5] (Figure 2). Using the protocol described above, we easily characterized the GM-CSF and type VIII collagen-expressing cell types in cryosections and paraffin-embedded samples of various arteries, in the myocardium, and in cultured vascular cells.
As shown in Figures 1 and 2, the protocol described represents an excellent, easy to use means to evaluate the spatial and temporal expression pattern of mRNAs in various tissues and cultured cells. The protocol allows one to simultaneously characterize the expressing cell types, as well as the localization and distribution of other proteins. The protocol is as sensitive as radioactive in situ hybridization. However, it is less time consuming [1, 2] and more precisely locates the expressing cells.

One critical factor for converting the protocol from radiolabeled probes to nonradioactively-labeled probes has been the use of the highly sensitive DIG-labeled cRNA probes. Together with the enhancement of sensitivity by the DIG system, the use of the related products such as the DIG RNA Labeling Kit or the DIG Nucleic Acid Detection Kit provides in situ hybridization of consistently excellent quality.


'"/>

Source:


Page: All 1 2 3 4 5 6

Related biology technology :

1. Simple, Sensitive, and Rapid Detection of FLAG -Tagged Fusion Proteins
2. A New PCR-based Mycoplasma Detection Method
3. HSVision Molecular Beacon Detection Module Rapidly Detects Herpes Simplex Virus DNA
4. Detection and Identification of Phosphorylation Sites in Proteins Using LC/MS/MS with Neutral Fragment Loss Mapping
5. Gene Expression Arrays: Highly Sensitive Detection of Expression Patterns with Improved Tools for Target Amplification
6. The DIG System Nonradioactive and Highly Sensitive Detection of Nucleic Acids
7. Quantitative Measurement of Cell Proliferation Using the BrdU ELISA: A Comparison Between Colorimetric and Chemiluminescent Detection
8. Quantification of Nucleosomes in Serum by the Cell Death Detection ELISAplus
9. A Further Step in Understanding Apoptosis Direct Detection of PARP Cleavage
10. In Situ Cell Death Detection Kit
11. Detection of antibody-stained cell surface and intracellular protein targets with the Agilent 2100 bioanalyzer
Post Your Comments:
(Date:4/24/2015)... 24, 2015 Leading Regenerative Veterinary ... its allogeneic stem-cell product development program and changes ... the corporate strategic direction. , “I am extremely ... stem cell development program has been kicked into ... manufacturing facility and hiring of our new Director ...
(Date:4/24/2015)... April 24, 2015 /PRNewswire/ - BioAmber Inc. (NYSE: ... as the supplier of bio-succinic acid used to ... for textile applications that Bayer MaterialScience has begun ... IMPRANIL ® eco, a series of bio-based ... as high as 65%.  IMPRANIL ® eco ...
(Date:4/24/2015)... /CNW Telbec/ - Mirego, leader in mobile strategies and solutions, ... mobile app extensions for the all new Apple Watch. Made ... OMSignal and MediaMiser, these extensions will reinvent the way users ... for wrist use.  Available today in ... customizable tactile smartwatch that makes for an excellent buddy for ...
(Date:4/24/2015)... 2015 PAREXEL International Corporation (NASDAQ: PRXL ... that Partnerships in Clinical Trials named PAREXEL,s Drew ... Innovator of the Year.  The honor recognizes Mr. Garty,s ... Perceptive MyTrials ® Data-Driven Monitoring solution. ... 2015 Partnership Awards in Boston , ...
Breaking Biology Technology:Vet-Stem, Inc. Meets New Milestones and Changes Name to VetStem Biopharma 2Bayer MaterialScience Names BioAmber As Its Supplier For New Product Line - Bio-succinic acid forms backbone of innovative new bio-based polyurethanes for textiles 2Bayer MaterialScience Names BioAmber As Its Supplier For New Product Line - Bio-succinic acid forms backbone of innovative new bio-based polyurethanes for textiles 3Bayer MaterialScience Names BioAmber As Its Supplier For New Product Line - Bio-succinic acid forms backbone of innovative new bio-based polyurethanes for textiles 4Familiprix, OMSignal and MediaMiser apps available today for Apple Watch 2PAREXEL Honored With 2015 Clinical Innovator Of The Year Award From Partnerships In Clinical Trials 2PAREXEL Honored With 2015 Clinical Innovator Of The Year Award From Partnerships In Clinical Trials 3PAREXEL Honored With 2015 Clinical Innovator Of The Year Award From Partnerships In Clinical Trials 4PAREXEL Honored With 2015 Clinical Innovator Of The Year Award From Partnerships In Clinical Trials 5
... Firmenich,s world leadership in the ... flavor ingredients, - New S2383 flavor enhancer may be used in ... healthcare products to reduce the need for sucralose, ... Inc. (Nasdaq: SNMX ), a leading company,focused on using proprietary ...
... BOSTON, Nov. 6 Solos Endoscopy, Inc. (Pink,Sheets: SNDY) ... a,significant purchase order for laparoscopic instruments from the St. ... client of Solos,Endoscopy,s for over 19 yrs and it ... Saint Mary,s Hospital has played a vital role in ...
... Inc. (NTI(R)) (Nasdaq: NTII ) today announced its ... first quarter of the,company,s fiscal year ending June 30, ... was $3.6 million, compared to $3.9 million for the,three ... million,for the first quarter of fiscal 2009 compared to ...
Cached Biology Technology:SENOMYX AND FIRMENICH TO COLLABORATE ON COMMERCIALIZATION OF S2383, A NOVEL ENHANCER OF THE HIGH-INTENSITY SWEETENER SUCRALOSE 2SENOMYX AND FIRMENICH TO COLLABORATE ON COMMERCIALIZATION OF S2383, A NOVEL ENHANCER OF THE HIGH-INTENSITY SWEETENER SUCRALOSE 3SENOMYX AND FIRMENICH TO COLLABORATE ON COMMERCIALIZATION OF S2383, A NOVEL ENHANCER OF THE HIGH-INTENSITY SWEETENER SUCRALOSE 4SENOMYX AND FIRMENICH TO COLLABORATE ON COMMERCIALIZATION OF S2383, A NOVEL ENHANCER OF THE HIGH-INTENSITY SWEETENER SUCRALOSE 5Solos Endoscopy, Inc. Receives Purchase Order from Connecticut Hospital 2Neurobiological Technologies, Inc. Reports First Quarter Fiscal Year 2009 Financial Results 2Neurobiological Technologies, Inc. Reports First Quarter Fiscal Year 2009 Financial Results 3Neurobiological Technologies, Inc. Reports First Quarter Fiscal Year 2009 Financial Results 4Neurobiological Technologies, Inc. Reports First Quarter Fiscal Year 2009 Financial Results 5
(Date:4/20/2015)... , April 20, 2015 The ... Glenbeigh Records Management (GRM), Ireland,s ... Dubai Islamic Bank. Having built up an impressive track record ... a leading player within the records management sector in ... to double is GCC staffbase and employ a further eight ...
(Date:4/14/2015)... -- NXT-ID, Inc. (NASDAQ: NXTD ) ("NXT-ID" or ... growing mobile commerce market, announces that its Wocket® smart ... Design Awards under the category  ,Product Design – Technology,. ... are part of a global multi-disciplinary awards program. The ... marketplace, industry and judging panel. Winners will be announced ...
(Date:4/10/2015)... Research and Markets ( http://www.researchandmarkets.com/research/cjk4gb/security ) has ... - NEC" report to their offering. ... to supply a range of IT security management and ... on the development of a Big Data and cloud ... Asia-Pacific region is a core ...
Breaking Biology News(10 mins):Glenbeigh Records Management Wins Contract With Dubai Islamic Bank 2Glenbeigh Records Management Wins Contract With Dubai Islamic Bank 3NXT-ID's Wocket Smart Wallet Nominated for 2015 New York Design Awards in 'Product Design- Technology' 2NXT-ID's Wocket Smart Wallet Nominated for 2015 New York Design Awards in 'Product Design- Technology' 3NEC Security Competitive Profile 2015 2
... After a year of studying up close the symbiotic relationship ... University biologist has advanced the scientific understanding of biological diversity. ... relationship between two or more organisms that can be parasitic ... diversity as predation and competition. The Proceedings ...
... , Feb. 5, 2013 Reportlinker.com ... is available in its catalogue: ... and Prognostic Biomarkers, 2013 ... Endothelial Growth Factor (VEGF), which plays an ...
... When it comes to gene sequencing and personalized medicine for ... proteins are relatively easy to target with drugs and plenty ... study, University of Michigan Comprehensive Cancer Center researchers assess the ... kinases are acting up in a particular tumor. They go ...
Cached Biology News:Pirate-like flies connect symbiosis to diversity 2VEGF Inhibition: Therapy-Indicating and Prognostic Biomarkers, 2013 2VEGF Inhibition: Therapy-Indicating and Prognostic Biomarkers, 2013 3VEGF Inhibition: Therapy-Indicating and Prognostic Biomarkers, 2013 4VEGF Inhibition: Therapy-Indicating and Prognostic Biomarkers, 2013 5Study finds potential to match tumors with known cancer drugs 2Study finds potential to match tumors with known cancer drugs 3
LECHNER AND LaVECK MEDIUMfor the clonal growth of normal human bronchial and other epithelial cells. Requires additives. With glutamine.Recommended storage condition:oC to 8oCIntended use(s):research...
...
... Quality ISO 9001:2000 Certified USDA ... Precedures Validated, SOP's Training AALAS ... for All Procedures GLP Documentation Upon Request ... stop shopping point for all your custom ...
... Materials for 15-20 enhancements when ... Western Blot Signal Enhancement System is designed to ... is an excellent tool for evaluating proteins with low ... to pre-treat membrane prior to blocking the membrane; 1x ...
Biology Products: