Skip to Main Content

INFORMATION FOR

TUNEL Staining

Word

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.

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

Notes:

1ml Eppendorf tube is recommended to save on reagents. MAKE SURE it is digoxygenin-dUTP (DigUTP). 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.Rehydration: 5 minute washes:

___ Methanol
___ 75% Methanol
___ 50% Methanol
___ 25% Methanol
___ 100% 1xSSC

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

PBS Washes:

___ 15 min in PBS
___ 15 min in PBS

TdT Buffer Incubation:

___ 1 hour in 500 µl 1X TdT Buffer at RT

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

Label Terminal Ends:

____ Add 500 µl 1X TdT Buffer
____ Add 1 µl of 15U/µl TdT enzyme per 100 µl buffer to make 150U/ml TdT
____ 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

Note:EDTA is a calcium chelator that stops the transferase reaction.

1 mM EDTA/PBS Washes at 65ºC:

___ 1 hour
___ 1 hour

PBS Washes at RT:

___ 1 hour
___ 1 hour
___ 1 hour
___ 1 hour

MAB Washes at RT:

___ 5 minutes
___ 5 minutes

Blocking:

___ 1 hour in 2% BMB blocking solution at RT

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

Antibody Incubation:

___ 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 0.1% 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

5 MAB Washes at RT:

___ 1 hour
___ 1 hour
___ 1 hour
___ 1 hour
___ 1 hour

AP Buffer Washes at RT:

___ 5 minutes
___ 5 minutes

Note:

AP buffer inhibits endogenous phosphatases.
Staining:

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

Note:

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

Stop Color Reaction:

___ Stop chromogenic reaction in quick MAB wash

Fix Stain:

___ Bouin's Fix O/N at RT

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

DAY FOUR

Reagents:

70% buffered Ethanol
Methanol

___ Multiple 10 minute washes in 70% buffered EtOH at RT
___ 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).

TUNELimage001