Tunel for embryos

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TUNEL FOR EMBRYOS from Yi-Chun Wu, Gillian Stanfield and Bob Horvitz
2X fix mix

1M KCL 800ul

5M NaCl 40ul

0.5M EGTA 26ul

1M spermine 32ul (Sigma, dissolved fresh)

1M Hepes pH6.5 75ul

water 1527ul

MeOH 2.5ml

1X Buffer

5x buffer* 20ul

10% TX-100 1ul

CoCl2 6ul

water 73ul


Tdt Reaction Mix

5x buffer 20ul

10% TX-100 1ul

CoCl2 6ul

1mM Fl-dUTP** 1.5ul

TdT enzyme 1ul

water 70.5


Embryo Fix

2X fix mix 500ul

20% paraformaldehyde 100ul

35% glutaraldehyde 16ul

* The notes I have say that TdT comes with 5x buffer and CoCl2. But this is an old protocol, so I am not sure if this is still the case... they were purchased from Boehringer Mannheim, but BM was bought out by someone (at least once) and I am not sure what company that is now.

**Fl-dUTP is 2 parts 1mM dUTP: 1 part 1mM FITC-dUTP (very important to dilute the labeled nucleotides).

Tris-Triton buffer is 100mM Tris pH 7.4, 1% Triton X-100, 1 mM EDTA
PBST is 1x PBS + 0.5% Triton X-100.
1) Male 20% paraformaldehyde. Spin down to make sure junk is removed.

2) Wash worms off plate and hypochlorite treat. After larvae and adults are dissolved, wash 3x in water. Meanwhile, make fix mix (above).

3) After 3rd wash, suspend worms in 400ul of water. Chill 5 minutes on ice.

4) Meanwhile, mix together embryo fix (above) and vortex to mix.

5) Add 400ul embryos to tube, invert to mix, immediately freeze in liquid nitrogen.

6) Thaw in room temp water bath (~2min)

7) Fix 25 min RT, rocking

8) Wash 5 minutes in 1ml Tris-Triton buffer, rocking

9) Wash 3 X 5 min in 1ml PBST, rocking

10) Transfer (a few ul of packed embryos if you make a large volume) to 0.5 ml tube.

11) Incubate in 25ul 1x buffer for 5 min. SPin down and remove buffer.

12) Add 25ul reaction mix. Incubate 2 hours, 37°C in the dark (NOt necessary to agitate. can use PCR machine and set to 4°C after 2 hours if you want to leave overnight)

13) Wash 3x 10 minutes in 0.5ml PBST (Add DAPI if desired to final wash)

14) Mount in Vectashield

Protocl uses a swinging eppendorf microfuge to better remove all the supe for each wash. This is not necessary.
Other labeled nucleotides work (rhodamine and DIG) but were not as well tested.
Good luck.

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