Technical Notes: Detection of Apoptosis with Double Labeling: TUNEL and Active Caspase-3

Panel A. Single-labeled TUNEL-positive cells contain dark-brown nuclei.
Panel B. Caspase-3 positive cells with red-stained cytoplasm.
Panel C. TUNEL/caspase-3 double-labeled cells with both dark- brown nuclei and red-stained cytoplasm.

Panels A, B, and C represent five micron sections of paraffin-embedded mouse thymus which were processed according to the protocol listed. Apoptotic mouse thymocytes were identified using the TACS TdT DAB Apoptosis Detection Kit (Catalog # 4810-30-K) and immunohistochemical labeling of active caspase-3 using a Human/Mouse Active Caspase-3 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF835).

Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) can identify DNA fragmentation, a characteristic of both apoptotic as well as necrotic cells. To discriminate apoptotic cells from necrotic cells, an additional method of apoptosis detection should be used. For example, immunoreactivity of active caspases within a cell, in addition to TUNEL staining, can indicate that a cell is apoptotic vs. necrotic. The following protocol demonstrates an application of apoptosis detection with this type of double labeling: TUNEL and immunohistochemical labeling of active caspase-3.

Staining Protocol:

(Note: This protocol contains modifications of the protocols found within the two specified kits. Steps 1-10 use reagents from the TdT DAB Apoptosis Detection Kit (Catalog # 4810-30-K),and steps 11-23 use reagents from the Cell and Tissue Staining Kit.

  1. Rehydrate cryostat or paraffin-embedded (after deparaffinization in xylenes) tissue sections for 10 minutes in PBS at room temperature.
  2. Wash slides in DNase-free water for 5 minutes at room temperature.
  3. Treat tissue sections with Proteinase K solution by incubating them in a humidity chamber for 30 minutes at room temperature.
  4. Wash slides twice for 5 minutes in DNase-free water at room temperature.
  5. Block endogenous peroxidase with H2O2 for 10 minutes at room temperature.
  6. Repeat step 4.
  7. Incubate tissue sections in TdT labeling buffer for 5 minutes at room temperature.
  8. Apply TdT labeling mixture and incubate tissue sections in a humidity chamber for 1 hour at 37 °C.
  9. Rinse and then incubate tissue sections with Stop Buffer for 5 minutes at room temperature.
  10. Wash slides in PBS for 5 minutes at room temperature.
  11. Incubate tissue sections with Streptavidin-HRP (Catalog # 4800-30-06) for 30 minutes at room temperature.
  12. Wash slides twice for 5 minutes in PBS at room temperature.
  13. Incubate tissue sections with chromogenic solution DAB with DAB enhancer for 3-8 minutes at room temperature. Monitor nuclei staining (dark brown color) under the microscope.
  14. Wash slides in PBS for 20 minutes at room temperature.
  15. Repeat step 5.
  16. Rinse slides in PBS and apply avidin-biotin blocking reagents.
  17. Repeat step 12.
  18. Incubate tissue sections with R&D Systems' Anti-Active Caspase-3 Antibody (5-15 µg/mL, Catalog # AF835) overnight at 2-8 °C.
  19. Wash slides three times for 15 minutes in PBS at room temperature.
  20. Incubate tissue sections with anti-rabbit secondary antibody for 30-60 minutes at room temperature.
  21. Repeat step 19.
  22. Incubate tissue sections with Streptavidin-HRP (Catalog # 4800-30-06) for 30 minutes at room temperature.
  23. Repeat step 19.
  24. Incubate tissue sections for 2-5 minutes with AEC Chromogen and monitor red color staining development under the microscope.
  25. Rinse slides with PBS, mount using Aqueous Mounting Medium and then dry the slides.