scholarly journals Combined in situ hybridization chain reaction and immunostaining to visualize gene expression in whole-mount Drosophila central nervous systems

2021 ◽  
Author(s):  
Julia C Duckhorn ◽  
Ian P Junker ◽  
Yun Ding ◽  
Troy R Shirangi
Keyword(s):  
Gene Expression ◽  
Nervous System ◽  
In Situ Hybridization ◽  
Hybridization Chain Reaction ◽  
Chain Reaction ◽  
Nervous Systems ◽  
Whole Mount ◽  
Central Nervous Systems ◽  
Mrna And Protein Expression

Methods to visualize gene expression in the Drosophila central nervous system are important in fly neurogenetic studies. In this chapter, we describe a detailed protocol that sequentially combines in situ hybridization chain reaction (HCR) and immunostaining to detect mRNA and protein expression in whole-mount Drosophila larval and adult central nervous systems. We demonstrate the application of in situ HCR in comparisons of nervous system gene expression between Drosophila species, and in the validation of single-cell RNA-Seq results in the fly nervous system. Our protocol provides a simple, robust, multiplexable, and relatively affordable means to quantitatively visualize gene expression in the nervous system of flies, facilitating its general use in fly neurogenetic studies.

HCR RNA-FISH protocol for the whole-mount brains of Drosophila and other insects v1

2021 ◽  
Author(s):  
Amanda A. G. Ferreira ◽  
Bogdan Sieriebriennikov ◽  
Hunter Whitbeck
Keyword(s):  
Drosophila Melanogaster ◽  
In Situ Hybridization ◽  
Fluorescent In Situ Hybridization ◽  
Hybridization Chain Reaction ◽  
Chain Reaction ◽  
Alexa Fluor ◽  
Rna Fish ◽  
Whole Mount

This is a protocol to perform RNA fluorescent in situ hybridization (RNA-FISH) using hybridization chain reaction (HCR) on whole-mount samples of the brains of the fly Drosophila melanogaster and other insects, e.g. the jumping ant Harpegnathos saltator. Probes and HCR reagents are purchased from Molecular Instruments. This protocol is loosely based on the "generic sample in solution" protocol published by Molecular Instruments. Our modifications include the description of fixation conditions, counterstaining by Hoechst, and altered washes. Additionally, we use larger concentrations of probes and hairpins following the protocol described by Younger, Herre et al. 2020. We have successfully employed this protocol to stain insect brains with up to 4 different probe sets simultaneously (hairpins conjugated with Alexa Fluor 488, 546, 496, and 647).

scholarly journals Multidimensional quantitative analysis of mRNA expression within intact vertebrate embryos

2017 ◽  
Author(s):  
Vikas Trivedi ◽  
Harry M.T. Choi ◽  
Scott E. Fraser ◽  
Niles A. Pierce
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Mrna Expression ◽  
Hybridization Chain Reaction ◽  
Chain Reaction ◽  
Developmental Gene Expression ◽  
Vertebrate Embryos ◽  
Anatomical Space ◽  
Subcellular Resolution

ABSTRACTFor decades, in situ hybridization methods have been essential tools for studies of vertebrate development and disease, as they enable qualitative analyses of mRNA expression in an anatomical context. Quantitative mRNA analyses typically sacrifice the anatomy, relying on embryo microdissection, dissociation, cell sorting, and/or homogenization. Here, we eliminate the tradeoff between quantitation and anatomical context, using multiplexed in situ hybridization chain reaction (HCR) to perform accurate and precise relative quantitation of mRNA expression with subcellular resolution within whole-mount vertebrate embryos. Gene expression can be queried in two directions: read-out from anatomical space to expression space reveals co-expression relationships in selected regions of the specimen; conversely, read-in from multidimensional expression space to anatomical space reveals those anatomical locations in which selected gene co-expression relationships occur. As we demonstrate by examining gene circuits underlying somitogenesis, quantitative read-out and read-in analyses provide the strengths of flow cytometry expression analyses, but by preserving subcellular anatomical context, they enable iterative bi-directional queries that open a new era for in situ hybridization.SUMMARYMultiplexed in situ hybridization chain reaction (HCR) enables quantitative multidimensional analyses of developmental gene expression with subcellular resolution in an anatomical context.

scholarly journals FISHing for chick genes: Triple-label whole-mount fluorescence in situ hybridization detects simultaneous and overlapping gene expression in avian embryos

2004 ◽  
Vol 229 (3) ◽  
pp. 651-657 ◽  
Author(s):  
Nathaniel Denkers ◽  
Pilar García-Villalba ◽  
Christopher K. Rodesch ◽  
Kandice R. Nielson ◽  
Teri Jo Mauch
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Avian Embryos ◽  
Overlapping Gene ◽  
Whole Mount

Demonstration of Epstein-Barr virus in primary central nervous system lymphomas by the polymerase chain reaction and in situ hybridization

1990 ◽  
Vol 21 (5) ◽  
pp. 545-550 ◽  
Author(s):  
Emilie Rouah ◽  
Beverly B. Rogers ◽  
Deborah R. Wilson ◽  
Joel B. Kirkpatrick ◽  
Gregory J. Buffone
Keyword(s):  
Central Nervous System ◽  
Polymerase Chain Reaction ◽  
Nervous System ◽  
In Situ Hybridization ◽  
Epstein Barr Virus ◽  
Chain Reaction ◽  
Barr Virus ◽  
Polymerase Chain ◽  
Epstein Barr

scholarly journals Gene expression analysis on sections of zebrafish regenerating fins reveals limitations in the whole-mount in situ hybridization method

2007 ◽  
Vol 237 (2) ◽  
pp. 417-425 ◽  
Author(s):  
A. Smith ◽  
J. Zhang ◽  
D. Guay ◽  
E. Quint ◽  
A. Johnson ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Hybridization Method ◽  
Whole Mount
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