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TUNEL Staining

TUNEL detects cells undergoing apoptosis. Please see the paper by Hensey and Gautier for the details. In our hands, we detect very few if any apoptotic cells during normal development in Xenopus tropicalis using this protocol. However, the protocol is effective in identifying apoptotic cells in embryos that are undergoing substantial apoptosis.

This protocol is also available as PDF or Word.

Protocol thanks to Nancy Hoo

DAY ONE
Reagents:
Terminal Deoxynucleotidyl Transferase (TdT)
  • Invitrogen Cat #: 10533-0651X TdT Buffer
  • For 500 µl, add 400 µl PBS to 100 µl 5X Buffer
Digoxygenin-dUTP
  • Roche Cat #: 11-093-088-910
PBS
1X SSC
Bleach (For 5ml)
  • 0.125 ml 20X SSC
  • 0.2 ml hydrogen peroxide
  • 4.5 ml distilled water
  • 0.25 formamide
Rehydration:
5 minute washes
o Methanol
o 75% Methanol
o 50% Methanol
o 25% Methanol
o   100% 1xSSC
o   100% 1xSSC

Bleach:
o 1-2 hours in bleach under direct light

PBS Washes:
o 15 min in PBS
o 15 min in PBS

TdT Buffer Incubation:
o 1 hour in 500 µl 1X TdT Buffer at RT

Label Terminal Ends:
o Add 500 µl 1X TdT Buffer
o Add 1 µl of 15U/µl TdT enzyme per 100 µl buffer to make 150U/ml TdT
o Add 0.1 µl of DdUTP per 100 µl buffer (DdUTP stock is 1000X)
Leave Overnight at Room Temperature 

DAY TWO
Reagents:
1 mM EDTA/PBS
Dilute .5 M EDTA with PBS
MAB
Anti-digoxygenin AP antibody
(Roche Cat #:11-093-274-910)
2% BMB Blocking Reagent in MAB

1 mM EDTA/PBS Washes at 65ºC:
o 1 hour
o 1 hour

PBS Washes at RT:
o 1 hour
o 1 hour
o 1 hour
o 1 hour

MAB Washes at RT:

o 5 minutes
o 5 minutes

  1ml Eppendorf tube is recommended to save on reagents.
MAKE SURE it is digoxygenin-dUTP (DdUTP). DNA gets labeled not RNA! 

The buffer supplied by the kit is very limited. To make 5x Buffer: - Berger (1987) Meth. Enz. 152, 339.
1. Titrate 1 M Cacodylic acid to pH 7.2 with KOH.
2. Equilibrate K+ Cacodylate with Chelex 100 (ion exchanger from BioRad) at RT for 5 min. Chelex removes metals that can stimulate endonucleases.
3. Filter.
4. Add in order: H2O, DTT, CoCl2 to a final conc. of 0.5 M cacodylate, 1 mM DTT, 10 mM CoCl2***If CoCl2 is added before DTT, a ppt will form. Final buffer is tinted pink.
 

 

 

 

 

 

 

 

 

 

 

 

 

 

TdT is an enzyme that catalyzes the binding of digoxygenin conjugated dUTP to the 3¢ end of DNA strands.

 

 

 

 

 

 

 

 

 

 

 


EDTA is a calcium chelator that stops the transferase reaction.


Blocking:
o 1 hour in 2% BMB blocking solution at RT

Antibody Incubation:
o Add 1/3000 dilution of anti-digoxygenin AP antibody in 2% BMB Blocking Reagent at 4˚C O/N

DAY THREE
Reagents:
Alkaline Phosphatase buffer (AP Buffer)
Contains 5 mM levamisol and .1% Tween
MAB
Bouin's Fixative
  • 14 ml saturated Picric Acid
  • 1ml glacial acetic acid
  • 5 ml formaldehyde
NBT 75 mg/ml 70% DMF

BCIP 50 mg/mlin 100% DMF



BMB is a blocking reagent to prevent non-specific antibody binding.

5 MAB Washes at RT:
o 1 hour
o 1 hour
o 1 hour
o 1 hour
o 1 hour

 

These washes are necessary to wash off excess antibody. The washes are flexible. One can do a quick wash then wash overnight at 4˚C.

AP Buffer Washes at RT:
o 5 minutes
o 5 minutes

AP buffer inhibits endogenous phosphatases.


Staining:
o Transfer into NBT/BCIP 4.5 µl NBT + 3.5 µl BCIP per ml of AP buffer 4oC – may only take a few minutes 

Stop Color Reaction:
o Stop chromogenic reaction in quick MAB wash

We use NBT/BCIP instead of BM purple. The reaction comes up quickly and BM Purple often turns different colors - pale blue to purple and is quite superficial. NBT/BCIP behaves better and stays purple and quite nuclear rather than diffuse.


Fix Stain:
o Bouin's Fix O/N at RT 


DAY FOUR
Reagents:

70% buffered Ethanol

Methanol

 

o   Multiple 10 minute washes in 70% buffered EtOH at RT

o   Multiple 5 minute washes in Methanol

 

Take pictures of uncleared tissue, or embryos can be dehydrated and cleared in BB:BA clearing agent (2 parts benzyl benzoate/1 part benzyl alcohol).

 

 


The change in pH stops the color reaction and the formaldehyde fixes the stain.