Step 1: Sample collection, dissection and fixation

We collect and fix genital tissues from fly pupae at particular time points during development:

  • We collect Drosophila melanogaster male puparia and incubate them at 25˚C prior to dissection.
  • We dissect pupal terminalia in phosphate buffered saline (PBS) at 28 hours or 48 hour after puparium formation and fix them in paraformaldehyde at room temperature.
  • After washing, we store our samples in ethanol at -20˚C.

Download our detailed dissection protocol.


Step 2: Probe design and synthesis

We design and synthesize antisense RNA probes that will bind messenger RNA for our target genes:

  • We design 200-300 base pair probes for the largest exon present in all annotated isoforms.
  • We use PCR to amplify genomic sequence with an added T7 RNA polymerase binding sequence, and we synthesize our probes using in vitro transcription.
  • We purify our probes using ethanol precipitation and resuspend them in 50% formamide after analysis by Nanodrop.

Download our detailed probe protocol.


Step 3: In situ hybridization

We visualize gene expression patterns by applying probes to our samples and binding an antibody conjugated to an enzyme that catalyzes dye deposition:

  • We use the InsituPro VSi from Intavis (locally nicknamed Artoo Situ) to carry out in situ hybridization, although the same experiments can be performed by hand.
  • We bind digoxigenin-labeled probes to target mRNA, and recognize those probes with an antibody conjugated to alkaline phosphatase.
  • When exposed to additional chemicals, alkaline phosphatase catalyses the deposition of a purple dye in regions where target mRNA is expressed.

Download our detailed protocol (robot) and (by hand).


Step 4: Mounting and imaging

We image our samples using light microscopy:

  • We mount samples on microscope slides coated with ploy-L-lysine to help orient them correctly.
  • We image samples at 20X magnification on a Leica DM 2000 with a Leica DFC540 C camera.
  • Each image was Z stack-compiled with the Leica Application Suite to allow for visualization of expression patterns at different depths in the sample.


University of Pittsburgh



Vincent, B.J.*, Rice, G.R.*, Wong, G.M., Glassford, W.J., Downs, K.I., Shastay, J.L., Charles-Obi, K., Natarajan, M., Gogol, M.M., Zeitlinger, J., Rebeiz, M., 2019. An atlas of transcription factors expressed in the Drosophila melanogaster pupal terminalia. bioRxiv 677260. doi:10.1101/677260

*Since Gavin and Ben contributed equally to this work, please cite it as Rice and Vincent, et al. or Vincent and Rice, et al. We encourage you to flip a coin in deciding between those possibilities

This work is generously supported by NIH grant GM107387