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Salient

Salient is an excellent design with a fresh approach for the ever-changing Web. Integrated with Gantry 5, it is infinitely customizable, incredibly powerful, and remarkably simple.

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DNA staining of fixed worm with DAPI or Propidium Iodide

 

Notes: all spin steps are done for 1 min. at 1600 rpm in a swing-out eppendorf centrifuge, unless noted otherwise. To adapt the protocol for higher throughput, it is possible to add fixative and PBST for rehydration less carefully. In that case, try to take off as much supernatant as possible, add the fixative or PBST and briefly vortex to ensure complete mixing.

 

Fixation

  • Wash animals off plates with PBST.
  • Wash several times with PBST to get rid of the bacteria.
  • Optional: leave worms in PBST for about 20 min. to allow them to empty their guts of bacteria.
  • Take off as much liquid as possible using a drawn out capillary.
  • Add 1 ml of carnoys solution in a drop wise fashion, mixing by flicking the tube after each drop.
  • Fix at least 1 hour, and preferably O/N.

Rehydration

  • Spin the worms and remove as much fixative as possible using a drawn out capillary.
  • Add 1 ml PBST in a drop wise fashion, mixing by flicking the tube after each drop.
  • Let animals rehydrate for at least an hour. Longer is better, and I usually do several hours.

Staining with DAPI

  • Spin the worms down, and resuspend in 100 µl PBST with 2 µg/ml DAPI.
  • After a few minutes, mount the worms on a slide in mounting medium.

Staining with Propidium Iodide

  • Spin the worms down, and resuspend in PBST with 200 µg/ml DNAse free RNAse.
  • Incubate at 37°C for 1 hour.
  • Wash with TBST.
  • Resuspend worms in PBST with 2 µg/ml Propidium Iodide (500 µg/ml stock in water).
  • Incubate at 37°C for 30 min.
  • Spin animals down, and mount them on a slide in mounting medium.

Solutions

Carnoys solution (5 ml)

  • 3.0 ml ethanol
  • 1.5 ml acetic acid
  • 0.5 ml chloroform

PBST

  • 1x PBS with 0.05% Tween-20

Mounting medium

  • 10% glycerol
  • 2.3% DBCO
  • 10% PBS