TUNEL PROCEDURE IN
BOVINE EMBRYOS
Fabiola Paula-Lopes, F. Dean Jousan,
Dept. of Animal
Sciences,
and
Lehrstuhl für
Molekulare Tierzucht und Biotechnologie, Ludwig-Maximilians-Universität
Müenchen (FPL)
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modifications underway – we are adding
details on use of Hoescht – the procedure works as written but we are
trying to improve
Materials
8%
(w/v) paraformaldehyde stock solution:
Dissolve 8 g of powdered paraformaldehyde in 100 ml water. Heat and stir
(55-60 C – do not go higher). Add a few drops of 2 N sodium hydroxide until
the solution clears. Make fresh each day.
Alternatively, 8% paraformaldehyde can be purchased from Electron Microscopy
Sciences as a custom formulation in 4 ml aliquots (cat. no.
15710-SP). Throw away whatever is not
used in one day.
4%
(v/v) paraformaldehyde: 1:1 solution of
8% paraformaldehyde stock solution and 0.2 M PBS. Make up on the day of use.
Microscope
slides: dip the slides in
DNase (50 U/ml) - The stock is RQ1-RNA free DNase, Promega cat. # 610A,
concentration 1U/µl. Dilute 10
µl DNase with 190 µl PBS/PVP0µl. Use
fresh.
RNase
(50µg/ml) - Use RNase A (heat treated),
QIAGEN,
Mounting
medium and antifade: ProLong Antifade
Kit (Molecular Probes P-7481)
TUNEL
reaction mixture (In Situ Cell Death Detection Kit, Fluorescein: Boehringer
Mannheim; Cat. No. 1684795): Remove 100 µl Label Solution from bottle 2 for two
negative controls. Add total volume of bottle 1 (50 µl) to the remaining 450 µl
Label Solution in bottle 2 to obtain 500 µl TUNEL reaction mixture. Mix well to
equilibrate components. Note: The TUNEL
reaction mixture should be prepared immediately before use and should not be
stored.
Hoescht
33342: Prepare Stock 1 by dissolving 25
mg Hoechst 33342 (Sigma B2261) in 2.5 ml of distilled water (10 mg/ml). Store
at 4 C. On the day of use, prepare Stock
2 by diluting 5 µl Stock 1 in 10 ml PBS containing 1 mg/ml polyvinylpyrrolidone
(PBS-PVP) to produce a 5 µg/ml solution.
The working solution is prepared by diluting 200 µl Stock 2 with 800 µl
PBS-PVP (final concentration = 1 µg/ml).
Propidium
iodide (PI): Prepare a 2.5 mg/ml stock
by dissolving PI (Sigma; catalog number P4170) in PBS. Store the stock at 4 C. Immediately before use, add 100 µl PI to 900
µl PBS/PVP and then add 50 µl of this diluted solution to 200 µl PBS/PVP to
obtain the final working concentration of
50 µg/ml.
Note: Too much PI can obscure the TUNEL labeling
and, if RNA is not completely removed, lead to excessive cytoplasmic
staining. It may be necessary to use
lower concentrations of PI, or shorter staining time to get good results. We
have used concentrations as low as 0.5 µg/ml PI with good results. If possible we recommend use of Hoescht 33342
instead of PI.
Procedure for Performing TUNEL
Reaction for Embryos in Solution (Preferred Method)
1) Remove
embryos from embryo culture medium (KSOM) and wash 3 times in 50 µl drops of
PBS-PVP (2 min for each wash) by transferring the embryos from drop to drop.
2) Fix
embryos in 50 µl drops of paraformaldehyde solution [4% (w/v) in PBS, pH 7.4]
for 1 h at room temperature. Drops may evaporate if incubation is
continued for longer than 1 h.
3) Wash the
embryos 3 times in a 50 µl drop PBS/ PVP by transferring the embryos from drop
to drop (2 min for each wash).
4) Store the
embryos at 4 °C in
4-well plates until the initiation of the TUNEL procedure (or proceed to Step 5
with TUNEL procedure). If embryos were
stored, wash the embryos again as described in Step 3.
5) Incubate
embryos in a 50 µl drop of permeabilization solution [0.5% (v/v) Triton X-100,
0.1% (w/v) sodium citrate] for 30 min at room temperature in a humidified box
(a plastic box with wet towels will do).
Use of a PAP pen or other hydrophobic pen may aid in forming the
permeabilization drop. Incubation in the
permeabilization solution too long may cause embryos to lyse.
6) FOR
PI: Prepare and label 4 dolphin-nosed
tubes. One can add PBS/PVP immediately
but don’t add DNase, RNase, propidium iodide
or TUNEL mixture until just before before use.
a. DNase (190
µl PBS/PVP + 10 µl DNase)
b. RNase A
(999 µl PBS/PVP + 1 µl RNase)
c. Prepare PI
as described above.
d. TUNEL
(empty for now)
7) Wash the embryos
as described in Step 3. For positive and negative controls go to Step 8. For samples, proceed to Step 9.
8) For positive and negative control embryos only
-
Incubate positive and negative control embryos with RNA free DNase (50 U/ml) at
37 °C for 1 h in the dark. Wash embryos as described in Step 3.
(Continue to Step 9a).
9) Incubate
embryos in 25 µl drops of the TUNEL reaction mixture for 1 hour at 37 °C in the dark
(place embryos in box covered with aluminum foil).
a. For positive and negative control embryos only - Incubate positive
control embryos as described in Step 9. Incubate negative controls in the absence of the enzyme terminal transferase (bottle
1), only with label solution from bottle 2. Continue to Step 10.
10) Wash the
slides as described in Step 3.
11) If using
PI, incubate embryos with RNase A (50 µg/ml) for 1 h at room temperature in the dark. This step can be omitted if Hoescht 33342 is
used to stain nuclei.
i.
NOTE: DO NOT WASH EMBRYOS AFTER THIS STEP!!!
12) Incubate
embryos in a 25-50 µl drop of Hoescht 33342 or PI for 15 min at room temperature in the dark.
13) Wash the
embryos 7-8 times in PBS/PVP as described in Step 3.
14) Add 2-3 µl
of mounting medium (w/ antifade) to a poly-L-lysine coated slide.
15) Transfer
the embryos (5 to 20 embryos per drop) and cover the sample with a cover slip.
16) Determine
the number of fluorescent nuclei. Blue (Hoescht) or red (PI) fluorescence
indicates non-apoptotic cells and greenish-blue or teal (Hoescht) or
green/yellow (PI) fluorescence indicates apoptotic cells.
HINT: If background fluorescence is a
problem, wash the embryos for a longer duration in Step 13. It is crucial that the non-specific PI is
washed away from embryos prior to mounting them.
Procedure for Performing TUNEL Reaction for Embryos Affixed to Microscope Slides
NOTE: The procedure
is written for PI but can also be used with Hoescht instead
1)
Remove
embryos from embryo culture medium (KSOM) and wash 4 times in 100 µl drop of
PBS + 1 mg/ml polyvinyl-pyrrolidone (PVP) by transfering the embryos from drop
to drop.
2)
Fix
embryos in 100 µl drop of paraformaldehyde solution [4% (w/v) in PBS, pH 7.4]
for 1 h at room temperature. Wash the embryos 3 times in 100 µl drop PBS/ PVP
by transfering the embryos from drop to drop. Transfer the embryos to a
poly-l-lysine coated slide and allow embryos to dry for 24 hours at room
temperature.
3)
Wash
the slides twice by dipping in a Coplin jar containing PBS/PVP (2 minutes
each).
4)
Incubate
in permeabilisation solution [0.5% (v/v) Triton X-100, 0.1% (w/v) sodium
citrate] for 30 min at room temperature. IF you go too long, the embryo
may lyse.
5)
Wash
the slides as described in step 3.
6)
Incubate
positive and negative control with DNase (50 U/ml) at 37C for 1 h.
7)
Wash
the slides as described in step 3.
8)
Dry
the area around the sample and add 50 µl of TUNEL reaction mixture. Incubate for
1 hour at 37 C in the dark. Incubate negative controls in the absence of the
enzyme terminal transferase (label solution from bottle 2).
9)
Wash
the slides as described in step 3.
10)
Incubate
the slides with RNase A (50 µg/ml) for 1 h at room temperature.
11)
Blot
the slides, dry the area around the sample and add propidium iodide (0.5 µg/ml)
for 1 h at room temperature.
12)
Wash
the slides 4 times in PBS/PVP (2 minutes each).
13)
Dry
the area around the sample, add 16 µl of mounting medium (w/ antifade) to the
slide and cover the sample with coverslip.
14)
Determine
the number of fluorescent nuclei. Red fluorescence indicates nonapoptotic cells
and green/yellow (PI) or
(Hoescht)fluorescence indicates apoptotic cells.
Confocal image of a bovine embryo heat shocked
at 41 C for 9 h
and subjected to the TUNEL procedure using PI.
Epifluorescent
image of a bovine embryo heat shocked at 41 C for 9 h
and subjected to the TUNEL procedure using
Hoescht 33342.
modified
© Fabiola Paula Lopes,
