Detection of RNAs by in situ hybridization (ISH) is a well-established technique that permits the study of specific RNA expression patterns in tissues; however, not all tissues are equally amenable to staining using the same procedure. Here we describe a protocol that combines whole-mount immunofluorescence (IF) and fluorescence in situ hybridization (FISH) for the simultaneous detection of specific RNA transcripts and proteins, greatly enhancing the spatial resolution of RNA expression in complex, intact fly tissues. To date, we have successfully used this protocol in adult testis, larval male gonads, adult intestine and Malpighian tubules. IF is conducted in RNase-free solutions, prior to the harsh conditions of FISH, in order to preserve protein antigenicity within dissected tissues. Separate protocols are described for mRNA and miRNA detection, which are based on robust digoxigenin (DIG) RNA and locked nucleic acid (LNA) probes, respectively. The combined IF-FISH procedure can be completed in 2 d for miRNA detection and 4 d for mRNA detection. Although optimized for Drosophila, this IF-FISH protocol should be adaptable to a wide variety of organisms, tissues, antibodies and probes, thus providing a reliable and simple means to compare RNA and protein abundance and localization.
Bibliographical noteFunding Information:
acknoWleDGMents We are grateful to E. Bach, T. Xie, D. Harrison, N. Sokol and P. Lasko for their generosity with reagents and fly stocks, and to C. Doe, R. Hans, G. Volohonsky, A. Pasquinelli and S. Aigner for guidance on methods used in this manuscript. We also acknowledge I. Lohmann and C. Millane for successfully testing the protocol on Drosophila (larval and adult) and Hydractinia, respectively. In addition, we apologize to those colleagues whose work could not be referenced directly owing to space constraints. This work was supported by the G. Harold and Leila Y. Mathers Charitable Foundation, the Ellison Medical Foundation, the Emerald Foundation, the American Federation for Aging Research and the US National Institutes of Health (D.L.J.).
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology (all)