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TUNEL procedure for embryos

TUNEL PROCEDURE IN BOVINE EMBRYOS

Fabiola Paula-Lopes, Zvi Roth, F. Dean Jousan, P. Soto, and P. J. Hansen
Dept. of Animal Sciences, University of Florida

More Techniques     |  P.J. Hansen Home Page  |   UF Animal Sciences Home Page


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 1:10 poly-l-lysine solution (Sigma P8920) for 2 minutes. Allow the slides to dry.

         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 l Label Solution from bottle 2 for two negative controls. Add total volume of bottle 1 (50 l) to the remaining 450 l Label Solution in bottle 2 to obtain 500 l TUNEL reaction mixture. Mix well to equilibrate components. Note: The TUNEL reaction mixture should be prepared immediately before use and should not be stored.

 

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 l drops of PBS + 1 mg/ml polyvinyl-pyrrolidone (PVP) (2 min for each wash) by transferring the embryos from drop to drop.

2.       Fix embryos in 50 l 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 l 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 l 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.       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 l PBS/PVP + 10 l DNase)

a.       RNase A (999 l PBS/PVP + 1 l RNase)

b.       To prepare propidium iodide (PI): add 50 l PI (2.5 mg/ml) to 450 l PBS/PVP (now named PIa), then add 25 l PIa + 475 l PBS/PVP to obtain final concentration (now named PI).

c.       TUNEL (empty)

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 50 l 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.    Incubate embryos with RNase A (50 g/ml) for 1 h at room temperature in the dark.

NOTE: DO NOT WASH EMBRYOS AFTER THIS STEP!!!

12.    Incubate embryos in a 50 l drop of PIa (0.5 g/ml) 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 l 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. Red fluorescence indicates non-apoptotic cells and green/yellow 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

1.       Remove embryos from embryo culture medium (KSOM) and wash 4 times in 100 l drop of PBS + 1 mg/ml polyvinyl-pyrrolidone (PVP) by transfering the embryos from drop to drop.

2.       Fix embryos in 100 l 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 l 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 l 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 g/ml) for 1 h at room temperature.

11.    Blot the slides, dry the area around the sample and add propidium iodide (0.5 g/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 l 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 fluorescence indicates apoptotic cells.


Confocal image of a bovine embryo heat shocked at 41 C for 9 h
and subjected to the TUNEL procedure described above.


modified 3-11-03

Fabiola Paula Lopes, Zvi Roth, Dean Jousan, Paolete Soto, and Peter J. Hansen