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P Granules Staining

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Freeze-Crack P granule Staining Protocol

1. Prepare humidity chamber: line large square plate with humid paper towels and place 4 5ml plastic pipets across chamber to support slides.

Place aluminum block in dry ice. Fill 1 coplin jar (if you have only 1-10 slides) or Wheaton dish (if you have 10-20 slides) with acetone, fill another with methanol, place both in non-frost free -20oC freezer.

2. Polylysine coat slides as follows:

- Use all 3-well of 3-well brown slides from cel-line.

- Put 50uls. of polylysine solution onto each well. Be sure to cover the entire well.

- Let sit for 5-10minutes.

- Wipe excess liquid off the well with a KimWipe.

- Bake slides in a 60C oven for about 15 minutes.

3. Wash worms off the plate in PBS into a 15ml conical plastic tube.

4. Spin the worms in clinical centrifuge up to 1700 rpm. Do not use brake. Aspirate supernatant. The worm pellet may be very loose so be careful not to disturb it.

If you are staining more than one set of worms at a time, be sure to rinse out the aspirator between each set of worms by aspirating clean water through the system between each set .

5. Transfer worms to an eppendorf tube.

6. If necessary, wash worms in PBS buffer. Spin and aspirate as in step 4.

7. Pipet 20-40 worms onto the well of the slide with a 50ul. capillary pipet or a yellow tip. Be sure to cover the entire surface of the well.

8. Allow the worms to settle and stick to the slide for about 1 minute.

9. Pipet off excess liquid, you should leave only about 5ul of liquid.

10. Put a coverslip (24mm X 50mm) on each well at right angle to the slide, so that the edge of the coverslip extends over the edge of the slide.

11. Put pressure on the coverslip with a pair of forceps to just burst the adults. Do this while watching through the microscope. Try not to completely squish the worms and break open the embryos.

12. Put the slide on the prefrozen aluminum block on dry ice and put a little pressure on the coverslip while the slide freezes. The slide can be left on dry ice at this point until all slides have been frozen.

13. Pop the coverslip off the slide and immediately put the slide in prechilled (-20C) 100% methanol. Put in -20oC freezer for 15 minutes.

14. Put the slides in in prechilled (-20C) 100% acetone for 10 minutes.

15. Wash the slides in PBT Buffer (1XPBS, 0.1% Triton, 0.1% BSA) 3 x 10 minutes.

16. Incubate slides in PBT for 30 minutes.

17. Wipe off the back of the slide and around the well carefully with a Kimwipe.

18. Put 30uls of OIC1D4 (1:3 dilution in PBT) on each well.

Cover well with parafilm coverslip

Incubate at 4oC overnight in sealed humidity chamber.

19. Wash the slides in PBT 3 x 10 minutes.

20. Wipe off the back of the slide and around the well carefully with a Kimwipe.

21. Put 30uls of FITC-GOAT ANTI MOUSE (1:50 dilution in PBT) with DAPI (1:100 dilution of 0.1mg/ml)on each well.

22. Incubate at room temperature for 2 hours.

23. Wash the slides in PBT 3 x 10 minutes.

24. Wipe off the back of the slide and around the well carefully with a Kimwipe. Add one drop of vectarstain to each well and cover with a coverslip.