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 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 H2 O         1 x 3 min     3.  Circle sections with PAP pen and return to diH2 O.

PRETREAT     1.  Incubate with Proteinase K at RT for 30 min.     2.  Wash with diH2O, 4 x 2 min.     3.  Incubate with 2% H2 O2 at RT for 10 min.     4.  Rinse with diH2 O.

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

POST HYBRIDIZATION     1.  Submerge slides in TB buffer at RT for 15 min.     2.  Rinse in diH2 O.     3.  Cover slides with 2% BSA at RT for 10 min.     4.  Rinse in diH2 O.     5.  Immerse slides in PBS at RT for 5 min.

DETECTION     1.  Incubate with diluted Extraavidin-peroxidase link for 30 min at 37o C.     2.  Wash well with a stream of diH2 O.     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 diH2 O.     7.  Counterstain with modified Harris' hematoxylin and blue with PBS.

COVERSLIP     1.  Air dry, then mount with CrystalMount.     2.  Bake at 65o C 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