A fast in situ hybridization chain reaction method in Drosophila embryos and ovaries

Abstract

The in situ hybridization chain reaction (isHCR) is a powerful method for visualizing mRNA in many species. We present a rapid isHCR method for Drosophila embryos and ovaries. Ethylene carbonate was added to the hybridization buffer to facilitate the hybridization reaction, and a modified short hairpin DNA was used in the amplification reaction; these modifications decreased the RNA staining time from 3 days to 1 day. This method is compatible with immunohistochemistry and can detect multiple mRNAs. The proposed method could significantly reduce staining time for Drosophila researchers using isHCR.

Keywords: Drosophila melanogaster; embryo; in situ HCR; in situ hybridization chain reaction; ovary; rapid staining.

Figure 1.

Visualization of piwi mRNA and Vasa protein by EC-isHCR and immunohistochemistry. EC-isHCR signals (green) and anti-Vasa protein signals (magenta) were detected in stage-16 embryos. Embryos were stained using probes for piwi mRNA (a), EGFP mRNA (b), and piwi mRNA with xylene, proteinase K (proK), and fixative treatments (c). Arrows indicate background signals in the midgut. Scale bar: 100 µm.

Figure 2.

Detection of mRNAs using EC-isHCR in Drosophila whole-mount embryos and adult ovaries. (a–h) Differential interference contrast (DIC) images and ec-isHCR signals are shown. (a, b) nos (green) and gcl (magenta) mRNAs were detected in stage-5 embryo. (b) High magnification view of the region boxed in (a). (c) Ubx (green) and en (magenta) mRNAs were detected in stage-11 embryo. (d) nos (green) and K10 (magenta) mRNAs were detected in stage-10 egg chamber of the adult ovary. (e-h) Stage-5 embryos were stained using probes for ovo mRNA (e, f), EGFP mRNA (g, h). (f, h) High magnification view of the region boxed in (e, g) respectively. Arrowheads indicate primordial germ cells. Scale bars: 100 µm (a, c, d, e, g) and 20 µm (b, f, h).

Figure 3.

Visualization of nos mRNA using 3-day staining and EC-isHCR in whole-mount Drosophila adult ovaries. (a-c) Differential interference contrast (DIC) images and EC-isHCR signals are shown. nos mRNA (green) was detected in stage-10 egg chambers of adult ovaries. (a) The egg chamber was stained using 3-day staining method, and the image was captured with optimized confocal laser-microscopy settings (low-power laser). (b) The egg chamber was stained using EC-isHCR, and the image was taken under the low-power laser settings. (c) The egg chamber was stained using EC-isHCR, and the image was captured with optimized confocal laser-microscopy settings (high-power laser). Scale bars: 100 µm.