I am trying to determine apoptosis in mouse liver tissues using the Elabscience TUNEL In Situ Apoptosis Kit (HRP-DAB Method) (Catalog no. E-CK-A331). In my first experiment, I was able to identify apoptosis in the tissues and get the ideal staining. Even though I did not change anything in the protocol, the next day, on my second try, I was unable to get staining in the tissues. What do you think could be the reason for this?
Dear İremnur Sarıalioğlu
As you know the TUNEL In Situ Apoptosis Kit (HRP-DAB Method) are very sensitive to detect apoptosis in a single cell. Therefore, one has to be very careful while performing the assay and make sure that you adhere company manual protocol to get the desired results.
As said, you got a good apoptosis signal in the first experiment and now its is not coming to your expectations. I am sure that something went wrong either with your protocol, exposure time, washing and HRP-DAB which is light sensitive. Hence, I suggest you to double check and run one small assay by using positive as well as as negative controls samples only and check whether apoptotic signal appears??.
I suggest you to recall whether the kits components were not kept at RT for longer time or if any mixing of components by using same tips may cause serious issue. Other factors like samples over-fixing, concentration of TdT enzyme, prolonged reaction time may also affect apoptotic signal.
Make sure that:
1. The washing is sufficient, otherwise it will
affect the enzyme activity (such as DNase I and TdT
Enzyme) subsequent experimental operations.
2. After washing the slides with PBS, please carefully blot the liquid around
the sample areas with blotting paper.
4. Keep the sample moist during the experiment to prevent the
failure of the experiment caused by dry slides.
5. Avoid repeated freezing and thawing of the Labeling
Solution and TdT enzyme. Stirring by vortex is not
recommended.
Best
Shail
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