scholarly journals Hybridization chain reaction enables a unified approach to multiplexed, quantitative, high-resolution immunohistochemistry and in situ hybridization

Development ◽  
2021 ◽  
Vol 148 (22) ◽  
Author(s):  
Maayan Schwarzkopf ◽  
Mike C. Liu ◽  
Samuel J. Schulte ◽  
Rachel Ives ◽  
Naeem Husain ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
High Resolution ◽  
Signal Amplification ◽  
Brain Slices ◽  
Hybridization Chain Reaction ◽  
Unified Framework ◽  
Chain Reaction ◽  
One Step ◽  
Rna Imaging

ABSTRACT RNA in situ hybridization based on the mechanism of the hybridization chain reaction (HCR) enables multiplexed, quantitative, high-resolution RNA imaging in highly autofluorescent samples, including whole-mount vertebrate embryos, thick brain slices and formalin-fixed paraffin-embedded tissue sections. Here, we extend the benefits of one-step, multiplexed, quantitative, isothermal, enzyme-free HCR signal amplification to immunohistochemistry, enabling accurate and precise protein relative quantitation with subcellular resolution in an anatomical context. Moreover, we provide a unified framework for simultaneous quantitative protein and RNA imaging with one-step HCR signal amplification performed for all target proteins and RNAs simultaneously.

scholarly journals Hybridization chain reaction enables a unified approach to multiplexed, quantitative, high-resolution immunohistochemistry and in situ hybridization

2021 ◽  
Author(s):  
Maayan Schwarzkopf ◽  
Mike C. Liu ◽  
Samuel J. Schulte ◽  
Rachel Ives ◽  
Naeem Husain ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
High Resolution ◽  
Signal Amplification ◽  
Brain Slices ◽  
Hybridization Chain Reaction ◽  
Unified Framework ◽  
Chain Reaction ◽  
Formalin Fixed Paraffin Embedded ◽  
Rna Imaging

RNA in situ hybridization (RNA-ISH) based on the mechanism of hybridization chain reaction (HCR) enables multiplexed, quantitative, high-resolution RNA imaging in highly autofluorescent samples including whole-mount vertebrate embryos, thick brain slices, and formalin-fixed paraffin-embedded (FFPE) tissue sections. Here, we extend the benefits of 1-step, multiplexed, quantitative, isothermal, enzyme-free HCR signal amplification to immunohistochemistry (IHC), enabling accurate and precise protein relative quantitation with subcellular resolution in an anatomical context. Moreover, we provide a unified framework for simultaneous quantitative protein and RNA imaging with 1-step HCR signal amplification performed for all target proteins and RNAs simultaneously.

scholarly journals Combined microRNA and mRNA detection in mammalian retinas by in situ hybridization chain reaction

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Pei Zhuang ◽  
Huanqing Zhang ◽  
Ryan M. Welchko ◽  
Robert C. Thompson ◽  
Shunbin Xu ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Hybridization Chain Reaction ◽  
Chain Reaction ◽  
Mrna Detection

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 Optimizing the hybridization chain reaction-fluorescence in situ hybridization (HCR-FISH) protocol for detection of microbes in sediments

2021 ◽  
Author(s):  
Zeyu Jia ◽  
Yijing Dong ◽  
Heng Xu ◽  
Fengping Wang
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
False Positive ◽  
Signal Intensity ◽  
Sample Pretreatment ◽  
Extraction Methods ◽  
Hybridization Chain Reaction ◽  
Chain Reaction ◽  
Widespread Application

AbstractFluorescence in situ hybridization (FISH) is a canonical tool commonly used in environmental microbiology research to visualize targeted cells. However, the problems of low signal intensity and false-positive signals impede its widespread application. Alternatively, the signal intensity can be amplified by incorporating Hybridization Chain Reaction (HCR) with FISH, while the specificity can be improved through protocol modification and proper counterstaining. Here we optimized the HCR-FISH protocol for studying microbes in environmental samples, particularly marine sediments. Firstly, five sets of HCR initiator/amplifier pairs were tested on the laboratory-cultured bacterium Escherichia coli and the archaeon Methanococcoides methylutens, and two sets displayed high hybridization efficiency and specificity. Secondly, we tried to find the best combination of sample pretreatment methods and HCR-FISH protocol for environmental sample analysis with the aim of producing less false positive signals. Various detachment methods, extraction methods and formulas of hybridization buffer were tested using sediment samples. Thirdly, an image processing method was developed to enhance the DAPI signal of microbial cells against that of abiotic particles, providing a reliable reference for FISH imaging. In summary, our optimized HCR-FISH protocol showed promise to serve as an addendum to traditional FISH for research on environmental microbes.

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