FISH-Flow, a protocol for the concurrent detection of mRNA and protein in single cells using fluorescence in situ hybridization and flow cytometry
Riccardo ArrigucciYuri BushkinFelix RadfordKarim LakehalPooja VirRichard PineDecember MartinJeremy SugarmanYanlin ZhaoGeorge YapAlfred LardizabalSanjay TyagiMaria Laura Gennaro
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In recent years, the development of in situ technologies has made good progress. In situ hybridization (ISH) has become an important tool and has enabled the pathologist to demonstrate infectious pathogens or mRNAs in tissue sections or cytospins without destruction of morphology, thus enabling the assignment of signals to individual cells or cell compartments (, , , , , , , , ).
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Improved methods for both in situ hybridization and in situ enzyme histochemistry were described. The procedures for both methods have been significantly simplified by omitting some unnecessary treatments and substituting the cumbersome and laborious techniques, and the reliability of in situ histochemistry was increased by reversing the operations of Block and Debrouwer's procedure. The improved steps are: Instead of the conventional fixation, a simplified FAA procedure by adding liquid nitrogen onto the embedded tissues during sectioning to ensure high quality of the sections;labeled DNA by randompriming, other than labeled RNA by transcription, was used as probes in hybridization, which was conducted in a moisturesaturated plastic chamber other than emerging in mineral oil. The improved procedure for tissue in situ histochemical study was that the GUS coloration was carried out before fixation, embedding and sectioning, which was different from the procedure as described by Block and Debrouwer.
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A rapid method for construction of oligonucleotide arrays on a glass surface, using a novel heterobifunctional reagent, N‐(2‐trifluoroethanesulfonatoethyl)‐N‐(methyl)‐triethoxysilylpropyl‐3‐amine (NTMTA), has been described. The heterobifunctional reagent, NTMTA, carries two different thermoreactive groups. The triethoxysilyl group on one end is specific towards silanol functions on the virgin glass surface, while the trifluoroethanesulfonyl (tresyl) group on the other end of the reagent reacts specifically with aminoalkyl‐ or mercaptoalkyl‐ functionalized oligonucleotides. Immobilization of oligonucleotides on a glass surface has been realized via two routes. In the first one (A), 5′‐ aminoalkyl‐ or mercaptoalkyl‐functionalized oligonucleotides were allowed to react with NTMTA to form a oligonucleotide‐triethoxysilyl conjugate which, in a subsequent reaction with unmodified (virgin) glass microslide, results in surface‐bound oligonucleotides. In the second route (B), the NTMTA reagent reacts first with a glass microslide whereby it generates trifluoroethanesulfonate ester functions on it, which in a subsequent step react with 5′‐aminoalkyl or mercaptoalkyl oligonucleotides to generate support‐bound oligonucleotides. Subsequently, the oligonucleotide arrays prepared by both routes were analyzed by hybridization experiments with complementary oligonucleotides. The constructed microarrays were successfully used in single and multiple nucleotide mismatch detection by hybridizing these with fluorescein‐labeled complementary oligonucleotides. Further more, the proposed method was compared with the existing methods with respect to immobilization efficiency of oligonucleotides.
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Probes for In Situ Hybridization Histochemistry (M.E. Lewis and F. Baldino, Jr.). Detection of Messenger RNAs by In Situ Hybridization with Nonradioactive Probes (B. Bloch). In Situ Hybridization in Cells and Tissue Sections: A Study of Myelin Gene Expression during CNS Myelination and Remyelination (C.A. Jordan). The Use of In Situ Hybridization Histochemistry to Study Gene Expression in Mouse Neurological Mutants (G.D. Frantz and A.J. Tobin). In Situ Hybridization as a Means of Studying the Role of Growth Factors, Oncogenes, and Proto-Oncogenes in the Nervous System (M. Murray). The Use of In Situ Transcription in the Study of Gene Expression (J. Eberwine, I. Zangger, R. Van Gelder, C. Evans, and L. Tecott). In Situ Hybridization Histochemistry at the Electron Microscopic Level (J.-J. Soghomonian). Quantitative Analysis of In Situ Hybridization Using Image Analysis (A.J. Smolen and P. Beaston-Wimmer). Appendix. Index.
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