ArticlePloS one2015
A method for combining RNAscope in situ hybridization with immunohistochemistry in thick free-floating brain sections and primary neuronal cultures.
Article in PloS one, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 72 papers.
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Who cites it
72 citing papers in PubMed, 103 citations in OpenAlex.
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- Increased expression of CXCL10 and CCL3 salivary gland chemokines in primary Sjögren's syndrome detected and systematically quantified using RNAscope®in situ hybridization.Clinical and experimental immunology · 2025Article
- Engineered AAV capsid transport mutants overcome transduction deficiencies in the aged CNS.Molecular therapy. Nucleic acids · 2024Article
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- Corticotropin Releasing Factor (CRF) Coexpression in GABAergic, Glutamatergic, and GABA/Glutamatergic Subpopulations in the Central Extended Amygdala and Ventral Pallidum of Young Male Primates.The Journal of neuroscience : the official journal of the Society for Neuroscience · 2022Article
- Microglia-derived CCL2 has a prime role in neocortex neuroinflammation.Fluids and barriers of the CNS · 2022Article
- Tyramide signal amplification coupled with multiple immunolabeling and RNAScope in situ hybridization in formaldehyde-fixed paraffin-embedded human fetal brain.Journal of anatomy · 2022Article
- Prolonged breastfeeding protects from obesity by hypothalamic action of hepatic FGF21.Nature metabolism · 2022Article
12 more citing papers are in PubMed but not listed here.
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Authors and funding
4 authors at 1 institution in 1 country.
Funding
Abstract
In situ hybridization (ISH) is an extremely useful tool for localizing gene expression and changes in expression to specific cell populations in tissue samples across numerous research fields. Typically, a research group will put forth significant effort to design, generate, validate and then utilize in situ probes in thin or ultrathin paraffin embedded tissue sections. While combining ISH and IHC is an established technique, the combination of RNAscope ISH, a commercially available ISH assay with single transcript sensitivity, and IHC in thick free-floating tissue sections has not been described. Here, we provide a protocol that combines RNAscope ISH with IHC in thick free-floating tissue sections from the brain and allows simultaneous co-localization of genes and proteins in individual cells. This approach works well with a number of ISH probes (e.g. small proline-rich repeat 1a, βIII-tubulin, tau, and β-actin) and IHC antibody stains (e.g. tyrosine hydroxylase, βIII-tubulin, NeuN, and glial fibrillary acidic protein) in rat brain sections. In addition, we provide examples of combining ISH-IHC dual staining in primary neuron cultures and double-ISH labeling in thick free-floating tissue sections from the brain. Finally, we highlight the ability of RNAscope to detect ectopic DNA in neurons transduced with viral vectors. RNAscope ISH is a commercially available technology that utilizes a branched or "tree" in situ method to obtain ultrasensitive, single transcript detection. Immunohistochemistry is a tried and true method for identifying specific protein in cell populations. The combination of a sensitive and versatile oligonucleotide detection method with an established and versatile protein assay is a significant advancement in studies using free-floating tissue sections.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.