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