scholarly journals Non-Specific Binding, a Limitation of the Immunofluorescence Method to Study Macrophages In Situ

Genes ◽  
2021 ◽  
Vol 12 (5) ◽  
pp. 649
Author(s):  
Emma Sicherre ◽  
Anne-Laure Favier ◽  
Diane Riccobono ◽  
Krisztina Nikovics
Keyword(s):  
In Situ Hybridization ◽  
Expression Pattern ◽  
Specific Binding ◽  
Pig Model ◽  
Hybridization Chain Reaction ◽  
Immunofluorescence Method ◽  
Chain Reaction

Advances in understanding tissue regenerative mechanisms require the characterization of in vivo macrophages as those play a fundamental role in this process. This characterization can be approached using the immuno-fluorescence method with widely studied and used pan-markers such as CD206 protein. This work investigated CD206 expression in an irradiated-muscle pig model using three different antibodies. Surprisingly, the expression pattern during immunodetection differed depending on the antibody origin and could give some false results. False results are rarely described in the literature, but this information is essential for scientists who need to characterize macrophages. In this context, we showed that in situ hybridization coupled with hybridization-chain-reaction detection (HCR) is an excellent alternative method to detect macrophages in situ.

Analysis of HIV-1 expression in vivo with in situ hybridization and the polymerase chain reaction

1992 ◽  
Vol 6 (3) ◽  
pp. 215-221 ◽  
Author(s):  
B. Delord ◽  
M. Ottmann ◽  
M.-H. Schrive ◽  
J.-M. Ragnaud ◽  
J.-M. Seigneurin ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
In Situ Hybridization ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Hiv 1

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.

scholarly journals Multiplexed In Situ Hybridization Using Hybridization Chain Reaction

Zebrafish ◽  
2014 ◽  
Vol 11 (5) ◽  
pp. 488-489 ◽  
Author(s):  
Harry M.T. Choi ◽  
Victor A. Beck ◽  
Niles A. Pierce
Keyword(s):  
In Situ Hybridization ◽  
Hybridization Chain Reaction ◽  
Chain Reaction

scholarly journals Isolation of PCV3 from Perinatal and Reproductive Cases of PCV3-Associated Disease and In Vivo Characterization of PCV3 Replication in CD/CD Growing Pigs

Viruses ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 219 ◽  
Author(s):  
Juan Mora-Díaz ◽  
Pablo Piñeyro ◽  
Huigang Shen ◽  
Kent Schwartz ◽  
Fabio Vannucci ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Virus Production ◽  
Growing Pigs ◽  
Porcine Circovirus ◽  
Experimental Conditions ◽  
Histological Lesion ◽  
In Vivo Characterization

Porcine circovirus 3 (PCV3) has been identified as a putative swine pathogen with a subset of infections resulting in stillborn and mummified fetuses, encephalitis and myocarditis in perinatal, and periarteritis in growing pigs. Three PCV3 isolates were isolated from weak-born piglets or elevated stillborn and mummified fetuses. Full-length genome sequences from different passages and isolates (PCV3a1 ISU27734, PCV3a2 ISU58312, PCV3c ISU44806) were determined using metagenomics sequencing. Virus production in cell culture was confirmed by qPCR, IFA, and in situ hybridization. In vivo replication of PCV3 was also demonstrated in CD/CD pigs (n = 8) under experimental conditions. Viremia, first detected at 7 dpi, was detected in all pigs by 28 dpi. IgM antibody response was detected between 7–14 dpi in 5/8 PCV3-inoculated pigs but no IgG seroconversion was detected throughout the study. Pigs presented histological lesion consistent with multi systemic inflammation characterized by myocarditis and systemic perivasculitis. Viral replication was confirmed in all tissues by in situ hybridization. Clinically, all animals were unremarkable throughout the study. Although the clinical relevance of PCV3 remains under debate, this is the first isolation of PCV3 from perinatal and reproductive cases of PCV3-associated disease and in vivo characterization of PCV3 infection in a CD/CD pig model.

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