Abstract
An essential part of the drug discovery and development process in the pharmaceutical industry is to provide a full characterization of cells expressing a given drug target and potential downstream markers in human tissues and in relevant preclinical animal species. This task is best solved by a combination of methods, including histological assessment of target protein and mRNA using immunohistochemistry (IHC) and in situ hybridization (ISH), respectively, as well as non-histology-based methods, such as fluorescence-activated cell sorting (FACS), and single-cell (SCS) or single-nuclei (SNS) sequencing. In reality, this work is often complicated by a combination of low target expression levels and a less than optimal availability of specific reagents for detection. In particular, the ability to specifically detect low-abundance receptor targets using IHC is notoriously difficult, due to a daunting lack of commercially available specific antibodies validated for use in IHC. In the absence of fully validated antibodies and protocols for IHC, the specific detection of target mRNA using ISH is often the only available histological method. A highly sensitive, nonradioactive, automated, and robust ISH method for use on formalin-fixed, paraffin-embedded (FFPE) tissue sections is presented for assessing histological localization of mRNA transcripts of lowly expressed genes.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Pyke C, Knudsen L (2013) The glucagon-like peptide-1 receptor--or not? Endocrinology 154:4–8
Reubi JC (2014) Strict rules are needed for validation of G-protein-coupled receptor immunohistochemical studies in human tissues. Endocrine 47:659–661
Drucker DJ (2016) Never waste a good crisis: confronting reproducibility in translational research. Cell Metab 24:348–360
Gautron L (2019) On the necessity of validating antibodies in the immunohistochemistry literature. Front Neuroanat 13:46
Yusta B, Baggio LL, Koehler J et al (2015) GLP-1R agonists modulate enteric immune responses through the intestinal intraepithelial lymphocyte GLP-1R. Diabetes 64:2537–2549
Popovic D, Koch B, Kueblbeck M et al (2018) Multivariate control of transcript to protein variability in single mammalian cells. Cell Syst 7:398–411
Heppner KM, Kirigiti M, Secher A et al (2015) Expression and distribution of glucagon-like peptide-1 receptor mRNA, protein and binding in the male nonhuman primate (Macaca mulatta) brain. Endocrinology 156:255–267
Jensen CB, Pyke C, Rasch MG et al (2018) Characterization of the glucagonlike peptide-1 receptor in male mouse brain using a novel antibody and in situ hybridization. Endocrinology 159:665–675
Campbell JN, Macosko EZ, Fenselau H et al (2017) A molecular census of arcuate hypothalamus and median eminence cell types. Nat Neurosci 20:484–496
Skene NG, Bryois J, Bakken TE et al (2018) Genetic identification of brain cell types underlying schizophrenia. Nat Genet 50:825–833
Plasschaert LW, Žilionis R, Choo-Wing R et al (2018) A single-cell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocyte. Nature 560:377–381
Gall JG, Pardue ML (1969) Formation and detection of RNA-DNA hybrid molecules in cytological preparations. Proc Natl Acad Sci U S A 63:378–383
Pyke C, Salo S, Ralfkiaer E et al (1995) Laminin-5 is a marker of invading cancer cells in some human carcinomas and is coexpressed with the receptor for urokinase plasminogen activator in budding cancer cells in colon adenocarcinomas. Cancer Res 55:4132–4139
Wang F, Flanagan J, Su N et al (2012) RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. J Mol Diagn 14:22–29
Lisowski AR, English ML, Opsahl AC et al (2001) Effect of the storage period of paraffin sections on the detection of mRNAs by in situ hybridization. J Histochem Cytochem 49:927–928
Christoffersen BØ, Skyggebjerg RB, Bugge A et al (2019) Long-acting CCK analogue NN9056 lowers food intake and body weight in obese Göttingen Minipigs. Int J Obes. https://doi.org/10.1038/s41366-019-0386-0
Pyke C, Heller RS, Kirk RK et al (2014) GLP-1 receptor localization in monkey and human tissue: novel distribution revealed with extensively validated monoclonal antibody. Endocrinology 155:1280–1290
Acknowledgments
The author would like to thank Pia G. Mortensen, Bettina Brandrup, and Joan H. Rasmussen at Novo Nordisk A/S for technical expertise.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Pyke, C. (2020). Automated ISH for Validated Histological Mapping of Lowly Expressed Genes. In: Nielsen, B.S., Jones, J. (eds) In Situ Hybridization Protocols . Methods in Molecular Biology, vol 2148. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0623-0_14
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0623-0_14
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0622-3
Online ISBN: 978-1-0716-0623-0
eBook Packages: Springer Protocols