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Programmed Cell Death In Situ Detection
Using Terminal deoxynucleotidyl Transferase (TdT)

Terminal deoxynucleotidyl Transferase-mediated dUTP nick end labeling (TUNEL) is an in situ method for detecting the 3'-OH ends of DNA exposed during the internucleosomal cleavage that occurs during apoptosis.  Incorporation of biotinylated dUTP allows detection by immunohistochemical procedures.  The labeled apoptotic cells may be visualized by light microscopy.

Reference:  Gavrieli  et al.,  J. of Cellular Bio.  119:493-501, 1992.


    1.  Heat to 70o C for 10 min or to 60o C for 30 min.
    2.  Immediately place slides in             xylene          2 x 5 min
                                                              96% EtOh    2 x 3 min
                                                              90% EtOh    1 x 3 min
                                                              80% EtOh    1 x 3 min
                                                              di H2O         1 x 3 min
    3.  Circle sections with PAP pen and return to diH2O.

    1.  Incubate with Proteinase K at RT for 30 min.
    2.  Wash with diH2O, 4 x 2 min.
    3.  Incubate with 2% H2O2 at RT for 10 min.
    4.  Rinse with diH2O.

    1.  Cover slides with TdT buffer, tap off.
    2.  Add TdT/dUTP solution.
    3.  Incubate in humid chamber at 37oC for 1 hour.

    1.  Submerge slides in TB buffer at RT for 15 min.
    2.  Rinse in diH2O.
    3.  Cover slides with 2% BSA at RT for 10 min.
    4.  Rinse in diH2O.
    5.  Immerse slides in PBS at RT for 5 min.

    1.  Incubate with diluted Extraavidin-peroxidase link for 30 min at 37oC.
    2.  Wash well with a stream of diH2O.
    3.  Immerse in PBS, blot off.
    4.  Mix AEC substrates and add to slide.
    5.  Develop colour at RT to desired intensity, approximately 3 min.
    6.  Rinse with diH2O.
    7.  Counterstain with modified Harris' hematoxylin and blue with PBS.

    1.  Air dry, then mount with CrystalMount.
    2.  Bake at 65oC for 15 - 45 min.



Proteinase K, 20 ug/ml
Dilute 1.35 D14.8 mg/ml STOCK in 999 D PK buffer

2% H2O2
Dilute 6.67 ml 30% STOCK in 100 ml diH2O

2% BSA
Dissolve 2 g of BSA in 100 ml diH2O

TdT/dUTP solution, (1:50/ 1:10)
prepare 75 D per slide:
    1.5 D TdT
    7.5 D dUTP in 66 D of TdT buffer

Extraavidin-Peroxidase (1:10)
prepare 100 D per slide
    10 D in 90 D of diH2O

AEC substrate
To 5 ml H2O, add 2 drops A
                         3 drops B
                         2 drops C, mix well.


TdT Buffer, 100 ml
30 mM Tris, pH 7.2                                    3 ml of 1M
140 mM Na Cacodylate                                2.24 g
1 mM cobalt chloride                                  1 ml of 100 mM

100 mM Cobalt chloride, 100 ml
2.379 g

10 X TB Buffer, 100 ml
3 M NaCl                                                     17.53 g
0.3 M Na citrate                                            8.823 g
    dilute to 1x before use.

Proteinase K buffer, 100 ml
50 mM Tris (8.0)                                           5 ml of 1 M stock
1 mM EDTA                                                  200 ul of 0.5 M stock


Proteinase K solution                       Boehringer Manneheim:1413-783

Biotin-16-dUTP                              Boehringer Manneheim:1093-070

Terminal transferase                       Gibco/BRL:8008SB

ExtraAvidin Peroxidase                   SIGMA:E2886

AEC Substrate kit                           Vector:SK-4200

Harelco Harris hemotoxylin              Baxter:57735-3

Crystalmount                                 BioMeda:M02

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