May 16, 2019
DABCO Mounting Slide Protocol for Drosophila melanogaster embryos
- 1University of California, Berkeley
Protocol Citation: Ashley Albright 2019. DABCO Mounting Slide Protocol for Drosophila melanogaster embryos. protocols.io https://dx.doi.org/10.17504/protocols.io.2yjgfun
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 16, 2019
Last Modified: May 16, 2019
Protocol Integer ID: 23275
Abstract
Protocol for mounting Drosophila melanogaster embryos stored in PBS or PBT onto slides for conventional fluorescence and/or confocal microscopy.
Adapted from:
Kosman, D., Mizutani, C.M., Lemons, D., Cox, W.G., McGinnis, E., and Bier, E. (2004)
Materials
MATERIALS
1X PBS (Phosphate-buffered saline )
Microscope slides
GlycerolMerck MilliporeSigma (Sigma-Aldrich)Catalog #G5516
14-Diazabicyclo[2.2.2]octaneMerck MilliporeSigma (Sigma-Aldrich)Catalog #D27802
clear nail polish
Safety warnings
Read SDS for DABCO prior to starting this protocol!
Make Mountant
Make Mountant
In light-shielded 50 mL conical (I cover mine in foil) add 1.25 g of DABCO crystals, 15 mL of 1X PBS, and 35 mL of glycerol.
Safety information
Read SDS for DABCO before starting.
Mix on rocking platform until the solution is homogeneous. Store at -20 °C .
Mount Slides
Mount Slides
Take off as much liquid as you can, resuspend embryos in an arbitrary amount of DABCO mountant (depends on how dilute you want the embryos to be, but generally a couple hundred ul). Allow the embryos to settle 01:00:00 to 03:00:00 at Room temperature or overnight at 4 °C before mounting. Embryos can be stored for months/years in microcentrifuge tubes in light-shielded boxes at -20 °C .
Transfer approximately 35 µL of embryos with using pipette tip cut with scissors onto a clean slide between two 22 x 22 mm coverslips approximately 0.5 cm apart in the middle of the slide. Cover with another 22 x 22 mm cover slip. Seal the edges with a quick-drying, transparent nail polish. Slides can be stored for a few weeks at 4 °C in the dark (or a few months at -20 °C ) .
Note
Keep in mind if you're DAPI staining, that tends to diffuse away. It is better to mount a fresh aliquot of embryos if samples are reanalyzed at a later date.
Analyze embryos by conventional fluorescence or confocal microscopy.