Speeding up Fly Staining

by Harald Zähringer, Labtimes 02/2012

Staining or labelling internal organs of flies or other model organisms is a tedious and tough business. A new vacuum-assisted staining protocol may ease the pain of repetitive and laborious staining procedures.

Both forward genetic screens, based on random mutagenesis, or targeted reverse genetic screens applying RNAi, rely on the analysis of detectible phenotypes. Some of them, for example, Drosophila mutants with visible alterations in bristle morphology or wing shape are pretty easy to recognise. Alterations in internal organs, however, are much harder to observe and screening of genes responsible for internal organ development or regulation usually requires laborious fixation and staining of tissues.

Adapted vacuum system

Since traditional staining procedures or staining machines are either cumbersome or expensive, Veit Riechmann’s group from the Department of Cell and Molecular ­Biology at the University of Heidelberg developed a simple and inexpensive vacuum assisted-staining system for high throughput staining of internal organs in Drosophila or zebrafish embryos (Berns et al., Dev. Genes. Evol., 2012, epub ahead of print). Riechmann’s team studies Drosophila oogenesis as a model for the regulation of cell polarity and cytoskeleton dynamics. Hence, RNAi screens and staining of ovaries to detect genes regulating oogenesis are daily routine in the Riechmann lab.

The vacuum-assisted staining system is based on a commercially available vacuum manifold connected to a vacuum pump. A special 96-well filter plate, which is also commercially available, is implemented into the manifold to aspire liquids from the wells through the filter into a Büchner flask by applying a vacuum. For staining of Drosophila ovaries with the Riechmann device, ovaries are first dissected under a microscope and dropped into the filter plate wells filled with Schneider’s insect media (idle plate wells are simply masked with parcel tape). The filled plates are then mounted into the manifold and the insect media is aspired by an applied vacuum. In the next step, buffers and staining solutions necessary for fixation and DAPI-staining of the ovaries, are added with a multichannel pipette followed by incubation on a rocking platform.

Subsequently, stained ovaries are transferred from the wells onto microscope slides. To this end, the plastic grid at the bottom of the used filter wells is cut with a scalpel and removed by hand. The laid-open filters are then extruded from the plate by pushing them through the plate well with a yellow pipetting tip. Finally, the filter is transferred to a microscope slide with forceps.

According to Berns et al., 96 samples can be fixed and stained within 30 minutes. Due to the time required for dissecting and mounting of the ovaries, which turned out to be the rate limiting steps, 36 samples may be processed per person and day. However, the vacuum assisted-staining protocol is not restricted to Drosophila or certain tissues. Riechmann’s team has also used it to stain various Drosophila internal organs, such as larval brains and wing discs as well as for staining of cells and tissues in zebrafish embryos.

Innokenty Woichansky, PhD student in the lab of Veit Riechmann and coauthor of the vaccum-assisted staining paper, pipettes a sample into the 96-well filter plate in front of him. Unused wells of the plate are covered by parcel tape (brown wrap). The vacuum manifold left to the filter plate is connected to the vacuum pump. Aspired liquids are collected in the Büchner flask shown behind the pump.

Last Changed: 10.11.2012