| 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.
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