TUNEL方法

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.

DEPARAFFINIZE 
    1.  Heat to 70^o C for 10 min or to 60^o 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.

PRETREAT 
    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.

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

POST HYBRIDIZATION 
    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.

DETECTION 
    1.  Incubate with diluted Extraavidin-peroxidase link for 30 min at 37^oC. 
    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.

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

SOLUTIONS

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. 
 

STOCK SOLUTIONS

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 
 

PRODUCTS AND SUPPLIERS 
 

Proteinase K solution                       Boehringer Manneheim:1413-783 
 

Biotin-16-dUTP                              Boehringer Manneheim:1093-070 
 

Terminal transferase                       Gibco/BRL:8008SB 
(TdT)

ExtraAvidin Peroxidase                   SIGMA:E2886 
 

AEC Substrate kit                           Vector:SK-4200 
 

Harelco Harris hemotoxylin              Baxter:57735-3 
 

Crystalmount                                 BioMeda:M02