| 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
上一篇：TUNEL Assay Protocol [Rosen Lab, Baylor College of Medicine] 下一篇：In Situ Apoptosis Detection by TUNEL labeling (Frozen Section) [Wilcox Lab ,Emor
共7页: 上一页 [1] 2 [3] [4] [5] [6] [7] 下一页 | 
