scholarly journals Optimization and evaluation of fluorescence in situ hybridization chain reaction in cleared fresh-frozen brain tissues

2021 ◽  
Author(s):  
Vivek Kumar ◽  
David M. Krolewski ◽  
Elaine K. Hebda-Bauer ◽  
Aram Parsegian ◽  
Brian Martin ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Hybridization Chain Reaction ◽  
Chain Reaction ◽  
Fresh Frozen ◽  
Brain Tissues

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 Optimization and quantitative evaluation of fluorescence in situ hybridization chain reaction in clarified fresh-frozen brain tissues

2019 ◽  
Author(s):  
Vivek Kumar ◽  
David M Krolewski ◽  
Elaine K Hebda-Bauer ◽  
Aram Parsegian ◽  
Brian Martin ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Hybridization Chain Reaction ◽  
Chain Reaction ◽  
Postmortem Human Brain ◽  
Neuronal Populations ◽  
Spatiotemporal Information ◽  
Fresh Frozen ◽  
Imaging Depth ◽  
Brain Tissues

AbstractTranscript labelling in intact tissues using in situ hybridization chain reaction has potential to provide vital spatiotemporal information for molecular characterization of heterogeneous neuronal populations. However, it remains relatively unexplored in fresh-frozen brain which provides more flexible utilization than perfused tissue. In the present study, we optimized the combination of in situ hybridization chain reaction in fresh-frozen rodent brains and then evaluated the uniformity of neuronal labelling between two clearing methods, CLARITY and iDISCO+. We found that CLARITY yielded higher signal-to-noise ratios but more limited imaging depth and required longer clearing times, whereas, iDISCO+ resulted in better tissue clearing, greater imaging depth and a more uniform labelling of larger samples. Based on these results, we used iDISCO+-cleared fresh-frozen rodent brains to further validate this combination and map the expression of a few genes of interest pertaining to mood disorders. We then examined the potential of in situ hybridization chain reaction to label transcripts in cleared postmortem human brain tissues. The combination failed to produce adequate mRNA labelling in postmortem human cortical slices but produced visually adequate labeling in the cerebellum tissues. We next, investigated the multiplexing ability of in situ hybridization chain reaction in cleared tissues which revealed inconsistent fluorescence output depending upon the fluorophore conjugated to the hairpins. Finally, we applied our optimized protocol to assess the effect of glucocorticoid receptor overexpression on basal somatostatin expression in the mouse cortex. The constitutive glucocorticoid receptor overexpression resulted in lower number density of somatostatin-expressing neurons compared to wild type. Overall, the combination of in situ hybridization chain reaction with clearing methods especially iDISCO+ may find broad application in the transcript analysis in rodent studies but its limited use in postmortem human tissues can be improved by further optimizations.

Combination of Cytology, Fluorescence In Situ Hybridization for Aneuploidy, and Reverse-Transcriptase Polymerase Chain Reaction for Human Mammaglobin/Mammaglobin B Expression Improves Diagnosis of Malignant Effusions

2004 ◽  
Vol 22 (3) ◽  
pp. 474-483 ◽  
Author(s):  
Michael Fiegl ◽  
Margot Haun ◽  
Anita Massoner ◽  
Jens Krugmann ◽  
Elisabeth Müller-Holzner ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
In Situ Hybridization ◽  
Tumor Cell ◽  
Fluorescence In Situ Hybridization ◽  
Cell Detection ◽  
Malignant Cells ◽  
Chain Reaction ◽  
Tumor Cell Detection ◽  
Polymerase Chain

Purpose The identification of malignant cells in effusions by conventional cytology is hampered by its limited sensitivity. The aim of this study was to improve tumor cell detection in effusions by molecular approaches. Materials and Methods A total of 157 effusions from patients with tumors and 72 effusions from patients without a history or evidence of malignancy were included in this study. All effusion specimens were evaluated in parallel by cytology, fluorescence in situ hybridization (FISH) for aneuploidy, and reverse-transcriptase polymerase chain reaction (RT-PCR) for expression of human mammaglobin (hMAM) and mammaglobin B (hMAM-B). Results In effusions from patients with tumors, the sensitivities of tumor cell detection by cytology, FISH, and hMAM and hMAM-B detection were 46.2%, 53.3%, 36.4%, and 57.7%, respectively. The corresponding specificities were 94.4%, 97.0%, 87.1%, and 88.6%. Notably, a high percentage of effusions containing malignant cells were in fact transudates, indicating the necessity for molecular diagnostic work-up of transudates collected from patients with tumors. Dependent on the tumor type, the use of appropriate marker combinations improved tumor cell detection in effusions significantly. By combining all four diagnostic tests, a positive test result indicating the presence of malignancy was achieved in 81.1%, with a fairly good specificity of 70.1%. Conclusion Molecular techniques are definitely useful to detect malignancy in cytologically negative effusions. Tumor cell detection in effusions can be significantly improved by FISH and PCR techniques applying appropriate molecular markers. This finding should help to improve tumor staging, prognostic assessment, and treatment monitoring.

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