Abstract
We describe the detailed methods of “immunoFISH” to analyze the expression level and the spatial localization of RNA transcripts and proteins on cultured cells and formal-fixed, paraffin-embedded (FFPE) tissue sections. On cultured cells, we detect specific transcripts using the Stellaris fluorescence in situ hybridization (FISH) probes labeled with fluorophores that target multiple regions along the desired transcripts and proteins combining the immunofluorescent staining. On FFPE tissue sections, we use the RNAscope FISH probes, modified branched DNA (bDNA) probes to amplify the RNA signals, followed by immunofluorescent staining for protein detection. The abundance, composition, and spatial distribution are determined by signals from fluorescently labeled proteins and individual transcripts of images acquired using high-resolution fluorescence microscopy.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Kwon S, Chin K, Nederlof M, Gray JW (2017) Quantitative, in situ analysis of mRNAs and proteins with subcellular resolution. Sci Rep 7(1):16459. https://doi.org/10.1038/s41598-017-16492-1
Matos LL, Trufelli DC, de Matos MG, da Silva Pinhal MA (2010) Immunohistochemistry as an important tool in biomarkers detection and clinical practice. Biomark Insights 5:9–20
Eng J, Thibault G, Luoh SW, Gray JW, Chang YH, Chin K (2020) Cyclic multiplexed-immunofluorescence (cmIF), a highly multiplexed method for single-cell analysis. Methods Mol Biol 2055:521–562. https://doi.org/10.1007/978-1-4939-9773-2_24
Kwon S (2013) Single-molecule fluorescence in situ hybridization: quantitative imaging of single RNA molecules. BMB Rep 46(2):65–72
Femino AM, Fay FS, Fogarty K, Singer RH (1998) Visualization of single RNA transcripts in situ. Science 280(5363):585–590
Raj A, van den Bogaard P, Rifkin SA, van Oudenaarden A, Tyagi S (2008) Imaging individual mRNA molecules using multiple singly labeled probes. Nat Methods 5(10):877–879
Raj A, Tyagi S (2010) Detection of individual endogenous RNA transcripts in situ using multiple singly labeled probes. Methods Enzymol 472:365–386
Ludyga N, Grunwald B, Azimzadeh O, Englert S, Hofler H, Tapio S, Aubele M (2012) Nucleic acids from long-term preserved FFPE tissues are suitable for downstream analyses. Virchows Arch 460(2):131–140. https://doi.org/10.1007/s00428-011-1184-9
Saka SK, Wang Y, Kishi JY, Zhu A, Zeng Y, Xie W, Kirli K, Yapp C, Cicconet M, Beliveau BJ, Lapan SW, Yin S, Lin M, Boyden ES, Kaeser PS, Pihan G, Church GM, Yin P (2019) Immuno-SABER enables highly multiplexed and amplified protein imaging in tissues. Nat Biotechnol 37(9):1080–1090. https://doi.org/10.1038/s41587-019-0207-y
Wang F, Flanagan J, Su N, Wang LC, Bui S, Nielson A, Wu X, Vo HT, Ma XJ, Luo Y (2012) RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. J Mol Diagn 14(1):22–29
Kochan J, Wawro M, Kasza A (2015) Simultaneous detection of mRNA and protein in single cells using immunofluorescence-combined single-molecule RNA FISH. BioTechniques 59(4):209–212, 214, 216 passim. https://doi.org/10.2144/000114340
Acknowledgments
We thank Dr. Joe Gray (OHSU, USA) for providing his substantial support and conceptual guidance on our immunoFISH research. This work was partially supported by OHSU SOM Faculty Innovation Fund Pilot Program (S.K.).
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
Kwon, S., Chin, K., Nederlof, M. (2020). Simultaneous Detection of RNAs and Proteins with Subcellular Resolution. In: Ørom, U. (eds) RNA-Chromatin Interactions. Methods in Molecular Biology, vol 2161. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0680-3_6
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0680-3_6
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0679-7
Online ISBN: 978-1-0716-0680-3
eBook Packages: Springer Protocols