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 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 of raphe serotonergic neurons in fixed tissue sections  v1

2021 ◽  
Author(s):  
Alex Buckley
Keyword(s):  
In Situ Hybridization ◽  
Brain Tissue ◽  
Mouse Brain ◽  
Fluorescent In Situ Hybridization ◽  
Hybridization Chain Reaction ◽  
Chain Reaction ◽  
Serotonergic Neurons ◽  
Fixed Tissue ◽  
Tissue Sections

This is an RNA fluorescent in-situ hybridization (FISH) protocol that utilizes hybridization chain reaction technology from Molecular Instruments. The protocol fluorescently labels different mRNAs (up to 4 different mRNAs) such that they become suitable for imaging. This protocol is designed specifically for fixed mouse brain tissue sections that contain raphe serotonergic neurons, but can be applied to other regions of the mouse brain as well.

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