A method for combining RNAscope in situ hybridization with immunohistochemistry in thick free-floating brain sections and primary neuronal cultures

PloS One
Tessa M GrabinskiNicholas M Kanaan

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 hi...Continue Reading

References

Jun 1, 1969·Proceedings of the National Academy of Sciences of the United States of America·J G Gall, M L Pardue
Apr 1, 1995·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·H M KerstensA G Hanselaar
Dec 5, 1997·Histochemistry and Cell Biology·P Komminoth, M Werner
Feb 20, 1999·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·E J SpeelP Komminoth
Apr 17, 2001·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·A N PlayerJ A Kolberg
Nov 8, 2001·Molecular Biotechnology·S C AndrasM R Davey
Feb 27, 2003·Diagnostic Molecular Pathology : the American Journal of Surgical Pathology, Part B·Xiang Qian, Ricardo V Lloyd
Sep 7, 2004·Expert Review of Molecular Diagnostics·Mohamed J E M F Mabruk
Aug 30, 2006·Methods in Enzymology·Georges Lutfalla, Gilles Uze
Apr 5, 2007·Nature Protocols·Stefanie Kaech, Gary Banker
Mar 12, 2009·Molecular Therapy : the Journal of the American Society of Gene Therapy·Fredric P ManfredssonRonald J Mandel
Nov 28, 2009·The Anatomical Record : Advances in Integrative Anatomy and Evolutionary Biology·Roland Pochet, Jean-Pierre Timmermans
Apr 6, 2011·Nature Methods·Shalev Itzkovitz, Alexander van Oudenaarden
Dec 15, 2011·The Journal of Molecular Diagnostics : JMD·Fay WangYuling Luo
May 30, 2012·Analytical Biochemistry·G Sharath ChandraM Manamohan

❮ Previous
Next ❯

Citations

Apr 1, 2016·Journal of Neuroscience Methods·Andrew KneynsbergNicholas M Kanaan
Apr 20, 2016·Advanced Drug Delivery Reviews·Dmitriy KhodakovDavid Yu Zhang
Feb 13, 2016·European Journal of Endocrinology·Felix HaglundAnders Höög
Jan 30, 2018·Frontiers in Neuroanatomy·Maria Fe LanfrancoSonia Villapol
May 1, 2018·Diabetes, Obesity & Metabolism·Josephine B HebsgaardPeter H Kvist
Jun 12, 2018·Biomicrofluidics·D Huber, G V Kaigala
Jan 30, 2019·Genes & Development·Kaitlin J BashamGary D Hammer
Nov 22, 2018·ILAR Journal·Lauren E HimmelKelli L Boyd
Dec 12, 2018·ILAR Journal·Famke AeffnerKristin Wilson
Jul 2, 2020·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·Henrik JungerZoltan G Laszik
Dec 3, 2016·Frontiers in Molecular Neuroscience·Tessa Grabinski, Nicholas M Kanaan
Jan 11, 2019·Frontiers in Neural Circuits·Tomas HökfeltSven Ove Ögren
Sep 26, 2019·Frontiers in Neuroscience·Bojana GladanacShadab A Rahman
Apr 30, 2019·Frontiers in Endocrinology·Lotte Bjerre Knudsen, Jesper Lau
Nov 23, 2020·Neurobiology of Disease·David J MarmionJeffrey H Kordower
Aug 4, 2019·Journal of Comparative Pathology·A OchiY Katayama
Jul 14, 2021·Chemical Reviews·Min LiQiang Xia
Jul 21, 2021·Acta Neuropathologica Communications·Amer ToutonjiStephen Tomlinson
Sep 1, 2021·Journal of Neuroendocrinology·Carly M DrzewieckiJanice M Juraska

❮ Previous
Next ❯

Methods Mentioned

BETA
PCR

Software Mentioned

RNAscope
QuantiGene
ImageJ

Related Concepts

Related Feeds

Aminoglycosides

Aminoglycoside is a medicinal and bacteriologic category of traditional Gram-negative antibacterial medications that inhibit protein synthesis and contain as a portion of the molecule an amino-modified glycoside. Discover the latest research on aminoglycoside here.

Cell-Type-Specific Viral Vectors

Viral vectors are used in biological research and therapy to deliver genetic material into cells. However, the efficiency of viral vectors varies depending on the cell type. Here is the latest research on cell-type-specific viral vectors.

Cell-Type Specific Viral Vectors

Viral vectors are used in biological research and therapy to deliver genetic material into cells. However, the efficiency of viral vectors varies depending on the cell type. Here is the latest research on cell-type-specific viral vectors.

AAV-based Gene Therapy

Adeno-associated virus (AAV)-based gene therapy is a biological vector that is being researched to be used as a potential therapeutic option. This gene therapy is designed to insert fragments of DNA into targeted cells to help treat diseases, such as hemophilia a. Discover the latest research on AAV-based gene therapy here.

Astrocytes

Astrocytes are glial cells that support the blood-brain barrier, facilitate neurotransmission, provide nutrients to neurons, and help repair damaged nervous tissues. Here is the latest research.

Aminoglycosides (ASM)

Aminoglycoside is a medicinal and bacteriologic category of traditional Gram-negative antibacterial medications that inhibit protein synthesis and contain as a portion of the molecule an amino-modified glycoside. Discover the latest research on aminoglycoside here.

Astrocytes & Neurodegeneration

Astrocytes are important for the health and function of the central nervous system. When these cells stop functioning properly, either through gain of function or loss of homeostatic controls, neurodegenerative diseases can occur. Here is the latest research on astrocytes and neurodegeneration.

Basal Ganglia

Basal Ganglia are a group of subcortical nuclei in the brain associated with control of voluntary motor movements, procedural and habit learning, emotion, and cognition. Here is the latest research.

Cell-Type-Specific Viral Vectors (ASM)

Viral vectors are used in biological research and therapy to deliver genetic material into cells. However, the efficiency of viral vectors varies depending on the cell type. Here is the latest research on cell-type-specific viral vectors.