A Novel Fluorogenic Substrate for Alkaline Phosphatase for the Use of Nucleic Acid Hybridization Histochemistry and Cytochemistry.

Kagiyama, Naoto; Fujita, Shin; Momiyama, Masayoshi; Kondoh, Yasumitsu; Nishiyauchi, Miho; Hori, Samuel H.

doi:10.1267/ahc.28.581

Cited by 8 publications

(5 citation statements)

References 43 publications

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“…Naphthol AS-MX has been localized to a DNA sequence in metaphase spreads and interphase nuclei (Speel et al 1992) but not to mRNA in cells or tissues or to nucleic acids bound on membranes. The alkaline phosphatase substrates HNPP (2-hydroxy-3-naphthoic acid-2 Ј -phenylanilide phosphate) and HBNP (3-hydroxy-N -2 Ј -bi-phenyl-2-naphthalenecarboxamide phosphate ester) have been used to detect DNA hybridization on filters (Kagiyama et al 1992(Kagiyama et al ,1995Fujita et al 1994). HNPP has also been used in combination with Fast Red TR to detect specific bacterial cells with rRNA-targeted oligonucleotide probes (Yamaguchi et al 1996) and to detect targets on chromosomes, but only after a 2-hr incubation with the azo dye-coupled substrate (Kagiyama et al 1995).…”

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confidence: 99%

“…The alkaline phosphatase substrates HNPP (2-hydroxy-3-naphthoic acid-2 Ј -phenylanilide phosphate) and HBNP (3-hydroxy-N -2 Ј -bi-phenyl-2-naphthalenecarboxamide phosphate ester) have been used to detect DNA hybridization on filters (Kagiyama et al 1992(Kagiyama et al ,1995Fujita et al 1994). HNPP has also been used in combination with Fast Red TR to detect specific bacterial cells with rRNA-targeted oligonucleotide probes (Yamaguchi et al 1996) and to detect targets on chromosomes, but only after a 2-hr incubation with the azo dye-coupled substrate (Kagiyama et al 1995). Biotinylated or fluorophore-labeled tyramides have been successfully used as substrates for horseradish peroxidase to detect genes on chromosome spreads (Kerstens et al 1995) but have not been used for in situ detection of mRNA or for detecting DNA hybridization on filter membranes.…”

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The ELF-97 Alkaline Phosphatase Substrate Provides a Bright, Photostable, Fluorescent Signal Amplification Method for FISH

Paragas¹,

Zhang²,

Haugland³

et al. 1997

J Histochem Cytochem.

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SUMMARYWe used the ELF-97 ( E nzyme-L abeled F luorescence) phosphatase substrate, 2-(5 Ј -chloro-2-phosphoryloxyphenyl)-6-chloro-4(3H)-quinazolinone, with alkaline phosphatase conjugates of streptavidin and appropriate antibodies to amplify signals from biotinylated and haptenylated hybridization probes. The dephosphorylated product, ELF-97 alcohol, is a bright yellow-green fluorescent precipitate optimally excited at ‫ف‬ 360 nm, with emission centered at ‫ف‬ 530 nm. This large Stokes shift allows ELF-97 signals to be easily distinguished from sample autofluorescence and signals arising from counterstains or other fluorophores. The ELF-97 precipitate was extremely photostable compared to fluorescein, allowing multiple photographic exposures of samples without significant signal intensity loss. For RNA in situ hybridization, labeling was specific and localized well to targets in cultured cells, tissue sections, and whole-mount zebrafish embryos. ELF-97 signals developed in seconds to minutes and were easily distinguished from pigmented tissues or cells, unlike those obtained using colorimetric substrates. We used the substrate with singly biotinylated short oligonucleotides to detect actin mRNA in MDCK cells and actin and ␤ -galactosidase mRNA in LacZ ϩ mouse fibroblasts. We also used a biotinylated cDNA, complementary to the mRNA encoded by the constant region of the T-cell receptor ␤ -chain, to specifically identify T-cells in mouse lymph node tissue sections. With digoxigenin-labeled probes, we detected several developmentally expressed mRNAs in whole-mount zebrafish embryos. Hybridization to centromere repeat regions in human metaphase and interphase chromosomes was also detected; ELF-97 signals were manyfold brighter than signals obtained with fluorescein conjugates. Finally, Southern blot hybridization using singly labeled oligonucleotide probes yielded a sensitivity similar to that obtained with radioactivity.( J Histochem Cytochem 45:345-357, 1997 )

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The ELF-97 Alkaline Phosphatase Substrate Provides a Bright, Photostable, Fluorescent Signal Amplification Method for FISH

Paragas¹,

Zhang²,

Haugland³

et al. 1997

J Histochem Cytochem.

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“…Southern blot hybridization and estimation of the relative amounts of ID elements. Southern blot analysis was carried out according to the method of Kagiyama et al(1995). Briefly, genomic DNAs digested with a restriction enzyme, EcoRI or Sau3AI, were electrophoresed in a 0.7% agarose gel, and transferred to a Hybond-N + membrane (Amersham).…”

Section: Methodsmentioning

confidence: 99%

Genomic organization and chromosomal distribution of rat ID elements.

et al. 2001

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Identifier (ID) elements are members of a family of short interspersed nuclear elements (SINEs) in rodents. We investigated the genomic organization and chromosomal distribution of the ID elements in the rat, mouse and Chinese hamster. Southern blot hybridization analysis revealed that the ID elements are widespread in the rat genome, but concentrated in the mouse and Chinese hamster genomes, and that the copy of ID elements in the rat is about 5 times and 50 times that in the mouse and Chinese hamster, respectively. FISH analysis showed that the ID elements are predominantly distributed in the R-band regions of rat chromosomes. In mouse and Chinese hamster chromosomes, no specific distribution pattern of the ID elements was found. Furthermore, we found a distinct group of derivative ID elements in the rat, which contain partially repeated ID core domains, by PCR amplification using an ID core sequence. Such derivatives were not found in either the mouse or Chinese hamster. These findings suggest that explosive amplification of the ID elements in the rat has been accompanied by the occurrence of derivative ID elements and a predominant localization to the R-band regions. Similar associations found in the Alu family, one of the human SINEs, allow us to speculate that the rat ID elements and the human Alu family have analogous functions in chromosomal organization.

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“…The latter solution, by contrast, can achieve high sensitivity by acting as a true substrate for the target enzyme, thus leading to significant catalytic signal amplification. The release of a solid‐state fluorophore into the medium from an enzyme‐responsive probe was recognized as highly attractive in the early nineties;7–9 more recent reports based on aggregation‐induced emission attest to its renewed interest 10. 11 The only commercial “precipitation‐based probe” (ELF97‐phosphate, Life Technologies)12, 13 relies on highly insoluble hydroxyphenyl‐quinazolinone ( 3 a , Scheme 1),14 and targets generic phosphatase activity 15.…”

Section: Introductionmentioning

confidence: 99%

Tagging Live Cells that Express Specific Peptidase Activity with Solid‐State Fluorescence

et al. 2014

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A three-component probe harnesses the extraordinary properties of a solid-state fluorophore for the detection of living cells exhibiting a particular peptidase activity. The off-on mode by which the probe operates, the bright fluorescence of the resulting precipitate, and the rapid response allow an exceptional signal-to-background ratio during microscopic imaging. A tertiary carbamate link between the spacer and phenolic fluorophore is at the heart of the probe's long-term stability. The degree of chlorination of the probe determines its response time and thus its suitability for live-cell analysis. Our probe also allows highly resolved localization of peptidase activity during gel analysis or on agar. In comparison, probes releasing soluble fluorophores demonstrate complete diffusion of the fluorescent signal. These results demonstrate the probe's potential for diverse biomedical applications, including high-fidelity flow cytometry and sensitive colony assays.

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A Novel Fluorogenic Substrate for Alkaline Phosphatase for the Use of Nucleic Acid Hybridization Histochemistry and Cytochemistry.

Cited by 8 publications

References 43 publications

The ELF-97 Alkaline Phosphatase Substrate Provides a Bright, Photostable, Fluorescent Signal Amplification Method for FISH

The ELF-97 Alkaline Phosphatase Substrate Provides a Bright, Photostable, Fluorescent Signal Amplification Method for FISH

Genomic organization and chromosomal distribution of rat ID elements.

Tagging Live Cells that Express Specific Peptidase Activity with Solid‐State Fluorescence

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