A Method for Combining RNAscope In Situ Hybridization with Immunohistochemistry in Thick Free-Floating Brain Sections and Primary Neuronal Cultures

Abstract Electroporation is a widely used non-viral technique for the delivery of molecules, including nucleic acids, into cells. Recently, electronic microsystems that miniaturize the electroporation machinery have been developed as a new tool for genetic manipulation of cells in vitro, by integrating metal microelectrodes in the culture substrate and enabling electroporation in-situ. We report that non-faradic SiO2 thin film-insulated microelectrodes can be used for reliable and spatially selective in-situ electroporation of mammalian cells. CHO-K1 and SH-SY5Y cell lines and primary neuronal cultures were electroporated by application of short and low amplitude voltage transients leading to cell electroporation by capacitive currents. We demonstrate reliable delivery of DNA plasmids and exogenous gene expression, accompanied by high spatial selectivity and cell viability. Finally, we show that SiO2 thin film-insulated microelectrodes support a double and serial transfection of the targeted cells.

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In situ electroporation of mammalian cells through SiO2 thin film capacitive microelectrodes

Scientific Reports ◽

10.1038/s41598-021-94620-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

M. Maschietto ◽

M. Dal Maschio ◽

S. Girardi ◽

S. Vassanelli

Keyword(s):

Thin Film ◽

Mammalian Cells ◽

Genetic Manipulation ◽

Neuronal Cultures ◽

Primary Neuronal Cultures ◽

Sio2 Thin Film ◽

Low Amplitude ◽

In Situ Electroporation

AbstractElectroporation is a widely used non-viral technique for the delivery of molecules, including nucleic acids, into cells. Recently, electronic microsystems that miniaturize the electroporation machinery have been developed as a new tool for genetic manipulation of cells in vitro, by integrating metal microelectrodes in the culture substrate and enabling electroporation in-situ. We report that non-faradic SiO2 thin film-insulated microelectrodes can be used for reliable and spatially selective in-situ electroporation of mammalian cells. CHO-K1 and SH-SY5Y cell lines and primary neuronal cultures were electroporated by application of short and low amplitude voltage transients leading to cell electroporation by capacitive currents. We demonstrate reliable delivery of DNA plasmids and exogenous gene expression, accompanied by high spatial selectivity and cell viability, even with differentiated neurons. Finally, we show that SiO2 thin film-insulated microelectrodes support a double and serial transfection of the targeted cells.

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

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100122885 ◽

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.

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Ultrastructural analysis of the spatial distribution of MRNA

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100123167 ◽

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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