TUNEL 方法

Terminal deoxynucleotidyl Transferase-mediated dUTP nick end labeling (TUNEL) is anin situmethod 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/mlDilute 1.35 D14.8 mg/ml STOCK in 999 D PK buffer

2% H2O2Dilute 6.67 ml 30% STOCK in 100 ml diH2O

2% BSADissolve 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 substrateTo 5 ml H2O, add 2 drops A 3 drops B 2 drops C, mix well.

STOCK SOLUTIONS

TdT Buffer , 100 ml30 mM Tris, pH 7.2 3 ml of 1M140 mM Na Cacodylate 2.24 g1 mM cobalt chloride 1 ml of 100 mM

100 mM Cobalt chloride, 100 ml2.379 g

10 X TB Buffer , 100 ml3 M NaCl 17.53 g0.3 M Na citrate 8.823 g dilute to 1x before use.