Description: This protocol is used for detection and quantification of apoptosis (programmed cell death) at single cell level, based on labeling of DNA strand breaks (TUNEL technology). Cleavage of genomic DNA during apoptosis may yield double stranded as well as single strand breaks (“nicks”), which can be identified by labeling free 3’-OH terminal with modified nucleotides in an enzymatic reaction.

Positive Controls: 1) Incubate sections with DNase I (3000U/ml in 50 mM Tris-HCl, pH 7.5, 1mg/ml BSA) for 10 minutes at 15-25 ºC to induce DNA strand breaks, prior to labeling procedure. 2) using known positive control is an alternative.

Negative Control: incubate sections with label solution only (without terminal transferase) instead of TUNEL reaction mixture.

Solutions and Reagents:

TdT Buffer Stock Solution (125mM Tris-HCl, 1M Sodium Cacodylate, 1.25mg/ml BSA, pH 6.6):

Tris-HCl (MW 157.6) ----------------------------------- 1.97 g

Sodium cacodylate, Trihydrate (MW 214.0) --------- 21.4 g

BSA ------------------------------------------------------- 0.125 g

Distilled water ------------------------------------------ 100 ml

Adjust pH to 6.6 and aliquot and store at –20 ºC.

Cobalt Chloride Stock Solution (25mM Cobalt Chloride in Distilled Water):

Cobalt chloride, Hexahydrate (MW 237.9) ------------ 0.6 g

Distilled water ------------------------------------------- 100 ml

Mix to dissolve. Aliquot and store at –20 ºC.

TdT Reaction Buffer (25mM Tris-HCl, 200 mM Sodium Cacodylate, 0.25 mg/ml BSA, 1mM Cobalt Chloride):

TdT Buffer Stock Solution ------------------------------ 40 ul

Cobalt Chloride Stock Solution ------------------------- 8 ul

Distilled water ------------------------------------------- 160 ul

Mix well. Store at –20 ºC

TdT Storage Buffer (60mM K-phosphate, pH 7.2, 150mM KCl, 1mM 2-Mercaptoethanol, 0.5% Triton X-100, 50% glycerol)

      To make the buffer:

      K2HPO4 (MW174.18) ------------------------------------ 1.05 g

      KCl (FW 74.55) ------------------------------------------- 1.12 g

      Distilled water -------------------------------------------- 50 ml

      Stir to dissolve and adjust pH 7.2 using concentrated HCl. Add 50 ml of glycerin (100% glycerol), 0.5 ml of Triton X-100, and 8 ul of 2-Mercaptoethanol (99% Solution. FW 78.13). Store at –20 ºC

Enzyme Reagent:

      Terminal Transferase (TdT) (Roche Diagnostic) ------ 4 ul

      TdT Storage Buffer -------------------------------------- 100 ul

      Mix well and store at –20 ºC

Label Reagent:

      Biotin-16-dUTP (Roche Diagnostic) --------------------- 4 ul

      TdT Reaction Buffer -------------------------------------- 1 ml

      Mix well and store at –20 ºC

TdT Reaction Mixture:

       Enzyme Reagent ----------------------------------------- 100 ul

       Label Reagent -------------------------------------------- 900 ul

       Mix just before use. The remaining 100 ul of Label Solution can be used for negative control.

            
Stop Wash Buffer (300mM NaCl, 30mM Sodium Citrate):

NaCl (MW 58.44) ----------------------------------------- 1.75 g

Sodium citrate, Trihydrate (MW294.11) --------------- 0.88 g

Distilled water -------------------------------------------- 100 ml

Mix to dissolve and store at room temperature.

Procedure:

1. Deparaffinize sections in 2 changes of xylene for 5 minutes each, and hydrate with 2 changes of 100% ethanol for 3 minutes each, and 95% ethanol for 1 minute.
2. Rinse in distilled water.
3. Pretreatment: Use proteinase K digestion method. Note: for frozen sections or culture cells grown on slides, incubate with 0.2% Triton X-100 in PBS-Tween for 30 minutes is required.
4. Rinse sections in 2 changes of PBS-Tween 20, 2 minutes each.
5. Peroxidase Blocking: incubate sections in 3% H2O2 in PBS for 10 minutes to block endogenous peroxidase activity.
6. Rinse in PBS-Tween 20 for 3x2 min.
7. Pre-incubation: incubate sections in TdT Reaction Buffer for 10 minutes.
8. TdT Reaction: incubate sections in TdT Reaction Mixture for 1-2 hours at 37-40 °C in humidified chamber.
9. Stop Reaction: rinse sections in stop wash buffer for 10 minutes.
10. Rinse in PBS-Tween 20 for 3x2min.
11. Detection: incubate sections with Streptavidin-HRP in PBS for 20 minutes at room temperature.
12. Rinse in PBS-Tween 20 for 3x2min.
13. Chromagen/Substrate: incubate sections with DAB for 1-2 minutes.
14. Rinse in tap water.
15. Counterstain with Gill's hematoxylin for 30 seconds.
16. Rinse in running tap water for 5 minutes.
17. Dehydrate through 95% ethanol for 5min, 100% ethanol for 2x3min.
18. Clear in xylene for 2x5min.
19. Coverslip with xylene based mounting medium.

Results:

Staining Pattern: Nuclear

References:

1. Gavrieli Y, et al (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol. 119:493-501.
2. Charriaut-Marlangue C and Ben-Ari Y (1995) A cautionary note on the use of TUNEL stain to determine apoptosis. NeuroReport 7:61-64.
3. Ay I, et al (2001) Intravenous basic fibroblast growth factor (bFGF) decreases DNA fragmentation and prevents downregulation of Bcl-2 expression in the ischemic brain following middle cerebral artery occlusion in rats. Molecular Brain Res. 87:71-80.