Abstract
The RNA in situ hybridization assay is essential in many studies to evaluate gene expression in vivo. It consists of generating tissue sections and subsequently hybridizing these sections with RNA probes. Keeping RNA intact is a challenge while harvesting tissue samples, processing through embedding, sectioning them, and conditioning the sections for hybridization. These challenges are particularly strong for adult skeletal tissues due to their copious, dense, and mineralized extracellular matrices. Here, we describe a method optimized to successfully hybridize RNA species, even of low abundance, in adult mouse bone and cartilage samples. This method involves tissue fixation with paraformaldehyde, demineralization with Morse’s solution and paraffin embedding, all of which can be completed in 4 days. Sections are then generated and hybridized using a 1-day standard protocol. Sections prepared using this method are compatible with immunostaining and standard staining procedures for skeletal tissues.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Cross M, Smith E, Hoy D, Nolte S, Ackerman I, Fransen M, Bridgett L, Williams S, Guillemin F, Hill CL, Laslett LL, Jones G, Cicuttini F, Osborne R, Vos T, Buchbinder R, Woolf A, March L (2014) The global burden of hip and knee osteoarthritis: estimates from the global burden of disease 2010 study. Ann Rheum Dis 73(7):1323–1330. https://doi.org/10.1136/annrheumdis-2013-204763
Wilkinson DG (1995) RNA detection using non-radioactive in situ hybridization. Curr Opin Biotechnol 6(1):20–23. https://doi.org/10.1016/0958-1669(95)80004-2
Moorman AF, De Boer PA, Ruijter JM, Hagoort J, Franco D, Lamers WH (2000) Radio-isotopic in situ hybridization on tissue sections. Practical aspects and quantification. Methods Mol Biol 137:97–115. https://doi.org/10.1385/1-59259-066-7:97
Joeng KS, Regan J, Long F (2014) Radioactive in situ hybridization to detect gene expression in skeletal tissue sections. Methods Mol Biol 1130:217–232. https://doi.org/10.1007/978-1-62703-989-5_16
Morse A (1945) Formic acid-sodium citrate decalcification and butyl alcohol dehydration of teeth and bones for sectioning in paraffin. J Dent Res 24:143–153
Shibata Y, Fujita S, Takahashi H, Yamaguchi A, Koji T (2000) Assessment of decalcifying protocols for detection of specific RNA by non-radioactive in situ hybridization in calcified tissues. Histochem Cell Biol 113(3):153–159. https://doi.org/10.1007/s004180050434
Acknowledgments
This work was supported by NIH grant AR072649 and AR068308 to V.L.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
de Charleroy, C., Haseeb, A., Lefebvre, V. (2021). Preparation of Adult Mouse Skeletal Tissue Sections for RNA In Situ Hybridization. In: Haqqi, T.M., Lefebvre, V. (eds) Chondrocytes. Methods in Molecular Biology, vol 2245. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1119-7_6
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1119-7_6
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1118-0
Online ISBN: 978-1-0716-1119-7
eBook Packages: Springer Protocols