Methods in Neurosciences: PCR in Neuroscience
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Offered with generous support from Promega Corporation. UW-Madison students and special students admitted to the course and wishing to receive 2 credits will enroll through the University registration system and will remit tuition fees directly to the University.
Documentation of current enrollment in an academic institution is required for confirmation of this special rate. Requirements Eligibility : Graduate students, biotechnology and pharmaceutical industry employees, postdoctoral candidates and principal investigators with an interest in neurosciences are encouraged to register. B Steps for the application of ISH in transgenic mice. C Steps for the combination of ISH and double immunofluorescence staining. D Steps for the combined retrograde tracing and ISH. National Center for Biotechnology Information , U.
Journal List Front Neurosci v. Front Neurosci. Published online May 2. Author information Article notes Copyright and License information Disclaimer. This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Neuroscience. Received Nov 15; Accepted Apr 6. The use, distribution or reproduction in other forums is permitted, provided the original author s and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice.
No use, distribution or reproduction is permitted which does not comply with these terms. This article has been cited by other articles in PMC. TIF K. TIF 1. Abstract In situ hybridization ISH is a powerful technique that is used to detect the localization of specific nucleic acid sequences for understanding the organization, regulation, and function of genes.
Introduction In situ hybridization ISH is a powerful technique, which identifies the localization of specific genes to understand the structure and function of RNAs. Materials and methods Animals Mice 8—12 weeks of age were group-housed under a h light-dark cycle — light and bred in the animal facilities at Wuhan National Laboratory for Optoelectronics. RNA isolation During the experiments, the solutions were treated with 0. Primer design The primers were designed using programs from the U.
Open in a separate window. Hybridization The RNase-free requirements were maintained up to post-hybridization steps. Immunofluorescence For immunofluorescent staining, brain sections were washed with 0. Results The protocol illustrates a simplified method for the preparation of RNA probes and applications combined with immunofluorescent staining and retrograde tracing in the central nervous system Supplementary Figure 1. Figure 1. Key points for the stabilization of ISH signals To identify the applicability of the probe, we detected the SST gene expression in the central lateral amygdala CeL , which is known to be abundantly distributed with SST neurons Li et al.
Figure 2. ISH applied in the transgenic reporter mouse Mouse lines are widely used to visualize specific cell populations in the brain by transgenic expression with fluorescent proteins Abe and Fujimori, Figure 3. Combination of ISH and double immunohistochemistry ISH has been usually used to study co-expression with specific molecules combined with immunohistochemistry Newton et al. Figure 4. Combination of ISH and retrograde tracing techniques The combination of ISH with retrograde tracing can offer important neural connection information, as mentioned in previous reports Le Moine, ; Conte-Perales et al.
Figure 5. Discussion and conclusions In the present study, we introduce a simplified protocol to prepare DIG-labeled RNA probes based on PCR amplification, and its various applications in neuroscience. Author contributions HL: designed the research; RH: carried out experiments and analyzed results; SY: designed the primers and analyzed sequencing data; ML offered the technical support.
Conflict of interest statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Footnotes Funding. Click here for additional data file. References Abe T. Reporter mouse lines for fluorescence imaging. Growth Differ. Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques.
Current Laboratory Methods in Neuroscience Research
The CCK-system underpins novelty-seeking behavior in the rat: gene expression and pharmacological analyses. Neuropeptides 42 , — Detection of two different mRNAs in a single section by dual in situ hybridization: a comparison between colorimetric and fluorescent detection.
Methods , — Alkaline fixation drastically improves the signal of in situ hybridization. Nucleic Acids Res.
Double and triple in situ hybridization for coexpression studies: combined fluorescent and chromogenic detection of neuropeptide Y NPY and serotonin receptor subtype mRNAs expressed at different abundance levels. Cell Biol. London: Springer; , 58— Pigment epithelium-derived growth factor: modulating adult neural stem cell self-renewal. Neuroanatomical tracing combined with in situ hybridization: analysis of gene expression patterns within brain circuits of interest. Methods , 28— A central amygdala-substantia innominata neural circuitry encodes aversive reinforcement signals. Cell Rep.
FISHing for chick genes: triple-label whole-mount fluorescence in situ hybridization detects simultaneous and overlapping gene expression in avian embryos. A procedure for in situ hybridization combined with retrograde labeling of neurons: application to the study of cell adhesion molecule expression in DiI-labeled rat pyramidal neurons.
Somatostatin and its receptors from fish to mammals. A fast and efficient polymerase chain reaction-based method for the preparation of in situ hybridization probes. Histopathology 61 , — Notching up neural stem cell homogeneity in homeostasis and disease. A gene expression atlas of the central nervous system based on bacterial artificial chromosomes. Nature , — Genetic labeling of neurons in mouse brain. Cold Spring Harb. Validating transcripts with probes and imaging technology. Methods 8 , S12—S9. Characterization of adult neural stem cells and their relation to brain tumors.
Cells Tissues Organs , — In situ hybridization: methods and applications. Non-radioactive labeling and detection of nucleic acids. The complete backfile of the full-text is available back to volume 1 from As of , videos are selectively being added to accompany the written methods. Contains detailed protocols and descriptions of biochemical and biophysical techniques for research in biological and molecular sciences.
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