DAPI Staining Protocol
Adapted from: Ross Francis
- 1x PBS
- 0.1% Tween PBS
- 0.01% Tween PBS
- Methanol (stored at -20°C)
- Dilute DAPI Solution
- 75% glycerol solution
- 2% Agar pads
- Eppendorf MiniSpin Plus
- 1.5 mL microcentrifuge tubes (VWR, 14231-062)
- Drawn Pasteur pipette
- Remove stock DAPI stain from freezer and place in the dark.
- Pick worms you wish to stain into a labeled microcentrifuge tube containing about 1 mL of 0.01 % Tween PBS. Flick tube to mix.
- Centrifuge samples for 1-2 minutes at 1-3 rpm. The higher the rpm and the longer it is spun, the better the worms will pellet. Dissected worms may not be spun higher than 1 rpm.
- Aspirate off as much solution as possible using a drawn Pasteur pipette. Allow a small volume of solution to remain so that you don’t suck out the worms by accident.
- Wash the worms by adding 1 mL of 0.01% Tween PBS. Centrifuge samples for 1-2 minutes at 1-3 rpms.
- Repeat the 2nd and 3rd steps 2 more times.
- Aspirate off as much one last time, and then add 1 mL of methanol (-20°C). Flick hard to mix (whole worms only). Immediately place the tube into a freezer at -20°C. Leave in for exactly 5 minutes.
- If more than one or two worms are clinging to the sides of the tube, fill to top with 0.1 % Tween PBS and mix by shaking up & down or flicking.
- Centrifuge samples for 1-2 minutes at 1-3 rpm.
- Aspirate off the solution, rinse with .1% Tween PBS, and centrifuge.
- Add .2 µl of stock DAPI solution. Flick to mix. Let sit for 5 minutes in the dark.
- Aspirate off the solution and add 1 mL of 0.1% Tween PBS. Centrifuge samples for 1-2 minutes at 1-3 rpm.
- Aspirate off the solution.
- Add 30 µl of the glycerol solution.
- Using the wide-bore pipette, gently pipette the solution and worms up and down several times. Then, deposit half of the worms on each of two agar pads.
- Use a drawn pipette to remove the excess solution from the pads before applying a cover slip to each pad.
*Notes for taking pictures: Analyzer IN, Flat black piece to mid-right side to first click from all the way in, Binocular NOT Confocal, Black knob for camera IN, Black wheel behind filters turned all the way up, Neutral density all the way out