Tunel:for Detection of Apoptotic Cells
For frozen sections or cells on coverslips
(TUNEL= terminal deoxynucleotidyl transferase dUTP nick ending labeling)
***Always have a control sample in which TdT is not added.
1. Use tissue or cells fixed 2% paraformaldehyde in PBS (20 minutes cells/~1 or 2 hrs tissue).
2. Incubate with .1% Triton x-100 in PBS for 20 minutes. Wash cells or tissue with PBS approximately three times.
Roche kit is used as follows:
3. Put on TUNEL mixture (minimal volume per section), place a coverslip over sample to spread the volume, and incubate in humid chamber at 37oC for 30 min.
**TUNEL mixture: (vortex after each addition)
45 microliters dH2O
Buffer 32 microliters-TdT buffer
CoCl2 20 microliters
TdT 1 microliters (negative control does not include TdT)
Biotin 1 microliter
Total volume: 99 ul. This is enough for several coverslips or tissue section.
4. Remove slides from incubator and wash off TUNEL mixture thoroughly with 1X PBS
5. Put on streptavidin secondary (488 or Cy3) for 30 minutes room temperature.
6. Wash 3 times with 1X PBS
7. At this point you can label with additional antibodies. Minimally, samples should be counterstained with Hoechst or other nuclear dye. Proceed as directed for cell or tissue labeling. Otherwise coverslip with Gelvatol.
Specific staining should be nuclear and co-localize with your nuclear counterstain. Non-specific staining will be cytoplasmic. Avoid imaging the edges of samples
Terminal Transferase, recombinant (Roche, #03333566001, 8000units, ~$144)