| 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.
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