Combined MicroRNA In Situ Hybridization and Immunohistochemical Detection of Protein Markers

2019 ◽  
pp. 271-286
Author(s):  
Boye Schnack Nielsen ◽  
Kim Holmstrøm
Keyword(s):  
In Situ Hybridization ◽  
Immunohistochemical Detection ◽  
Protein Markers

Spectral Imaging of Chromogenic Dyes in Cytological Specimens

1997 ◽  
Vol 3 (S2) ◽  
pp. 143-144
Author(s):  
Merryn Macville ◽  
Ernst-Jan Speel ◽  
Dirk Soenksen ◽  
Ton Hopman ◽  
ThomasRied
Keyword(s):  
In Situ Hybridization ◽  
Spectral Imaging ◽  
Immunohistochemical Detection ◽  
Bright Field ◽  
Transmission Microscopy ◽  
Cytochemical Analysis ◽  
Unique Material ◽  
Bright Field Microscopy ◽  
Auto Fluorescence

Recently, spectral imaging has been successfully applied to 24-color fluorescence in situ hybridization (FISH), and it has evolved into a new powerful method for the analysis of structural and numerical chromosomal aberrations on metaphase spreads (spectral karyotyping) For cytological specimens it is often impossible to use fluorescence microscopy because of (fixation-induced) auto-fluorescence or prior cytological staining. Also, re-examination of archived fluorescent specimens is hampered by fading. However, the need for multi-parameter cytochemical analysis remains when rare or unique material is to be studied in research or clinical diagnosis. Multi-color bright-field microscopy using enzyme precipitates has become feasible for in situ hybridization as well as immunohistochemical detection. Conventional transmission microscopy optics have allowed up to three targets to be detected simultaneously. In parallel to 24-color FISH, we explored the potential of spectral imaging to increase the number of parameters to be analyzed in bright-field microscopy.

scholarly journals Immunohistochemical detection of FGF-23 protein in tumors that cause oncogenic osteomalacia

2003 ◽  
pp. 269-276 ◽  
Author(s):  
T Larsson ◽  
R Zahradnik ◽  
J Lavigne ◽  
O Ljunggren ◽  
H Juppner ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Polyclonal Antibodies ◽  
Affinity Purification ◽  
Fibroblast Growth Factor 23 ◽  
Immunohistochemical Analysis ◽  
Current Evidence ◽  
Immunohistochemical Detection ◽  
Mesenchymal Tumors ◽  
Fgf 23

OBJECTIVE: Oncogenic hypophosphatemic osteomalacia (OOM) is a rare disease characterized by hypophosphatemia, inappropriately low levels of circulating 1,25-dihydroxyvitamin D(3) and osteomalacia. The disease is most commonly caused by benign mesenchymal tumors that produce, among several other factors, fibroblast growth factor-23 (FGF-23). Current evidence thus suggests that this protein has an important role in the regulation of phosphate homeostasis. By producing polyclonal antibodies against human FGF-23 protein we wanted to determine the localization of FGF-23 protein in OOM tumors that express FGF-23 mRNA. DESIGN AND METHODS: Three polyclonal antibodies were raised in rabbits against three different peptides with sequences derived from human FGF-23: [Cys-70]FGF-23(51-69)amide, [Tyr-223]FGF-23(206-222)amide and [Tyr-224]FGF-23(225-244)amide. One of the resulting antisera was subsequently used for immunohistochemistry on sections from five different tumors causing OOM. FGF-23 mRNA expression was confirmed with in situ hybridization. RESULTS: After affinity purification, two of three antisera detected recombinant human FGF-23 by Western blot analysis. Through immunohistochemical analysis using the anti-[Tyr-224]FGF-23(225-244)amide antibody and through in situ hybridization using full-length antisense FGF-23 cRNA as a probe, we showed that abundant amounts of FGF-23 protein and mRNA are present in certain tumor cells of five different OOM tumors. CONCLUSIONS: We conclude that OOM tumors express FGF-23 protein and that the immunohistochemical detection of FGF-23 in OOM tumors is feasible and may help in establishing the diagnosis of tumor-induced hypophosphatemia through analysis of biopsies or surgical specimens.

DNA sequence mapping in interphase and metaphase chromosomes by fluorescence in situ hybridization

1992 ◽  
Vol 50 (1) ◽  
pp. 496-497
Author(s):  
Barbara Trask ◽  
Susan Allen ◽  
Anne Bergmann ◽  
Mari Christensen ◽  
Anne Fertitta ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Dual Band ◽  
Nick Translation ◽  
Metaphase Chromosomes ◽  
Band Pass ◽  
Texas Red ◽  
Fluorescent Spot

Using fluorescence in situ hybridization (FISH), the positions of DNA sequences can be discretely marked with a fluorescent spot. The efficiency of marking DNA sequences of the size cloned in cosmids is 90-95%, and the fluorescent spots produced after FISH are ≈0.3 μm in diameter. Sites of two sequences can be distinguished using two-color FISH. Different reporter molecules, such as biotin or digoxigenin, are incorporated into DNA sequence probes by nick translation. These reporter molecules are labeled after hybridization with different fluorochromes, e.g., FITC and Texas Red. The development of dual band pass filters (Chromatechnology) allows these fluorochromes to be photographed simultaneously without registration shift.

Ultrastructural analysis of the spatial distribution of MRNA

1992 ◽  
Vol 50 (1) ◽  
pp. 552-553
Author(s):  
Gary Bassell ◽  
Robert H. Singer
Keyword(s):  
Electron Microscopy ◽  
Spatial Distribution ◽  
In Situ Hybridization ◽  
High Resolution ◽  
Nucleic Acids ◽  
Colloidal Gold ◽  
Dna Probe ◽  
Nucleotide Analogs ◽  
Gold Particles

We have been investigating the spatial distribution of nucleic acids intracellularly using in situ hybridization. The use of non-isotopic nucleotide analogs incorporated into the DNA probe allows the detection of the probe at its site of hybridization within the cell. This approach therefore is compatible with the high resolution available by electron microscopy. Biotinated or digoxigenated probe can be detected by antibodies conjugated to colloidal gold. Because mRNA serves as a template for the probe fragments, the colloidal gold particles are detected as arrays which allow it to be unequivocally distinguished from background.

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