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.
DEPARAFFINIZE1. Heat to 70o C for 10 min or to 60o C for 30 min.2. Immediately place slides in xylene 2 x 5 min96% EtOh 2 x 3 min90% EtOh 1 x 3 min80% EtOh 1 x 3 mindi H2O 1 x 3 min3. Circle sections with PAP pen and return to diH2O.
PRETREAT1. 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.
HYBRIDIZE1. Cover slides with TdT buffer, tap off.2. Add TdT/dUTP solution.3. Incubate in humid chamber at 37oC for 1 hour.
POST HYBRIDIZATION1. 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.
DETECTION1. 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.
COVERSLIP1. Air dry, then mount with CrystalMount.2. Bake at 65oC for 15 - 45 min.
SOLUTIONSProteinase K, 20 ug/mlDilute 1.35 D14.8 mg/ml STOCK in 999 D PK buffer2% H2O2Dilute 6.67 ml 30% STOCK in 100 ml diH2O2% BSADissolve 2 g of BSA in 100 ml diH2OTdT/dUTP solution, (1:50/ 1:10)prepare 75 D per slide:1.5 D TdT7.5 D dUTP in 66 D of TdT bufferExtraavidin-Peroxidase (1:10)prepare 100 D per slide10 D in 90 D of diH2OAEC substrateTo 5 ml H2O, add 2 drops A3 drops B2 drops C, mix well.
STOCK SOLUTIONSTdT 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 mM100 mM Cobalt chloride, 100 ml2.379 g10 X TB Buffer, 100 ml3 M NaCl 17.53 g0.3 M Na citrate 8.823 gdilute to 1x before use.Proteinase K buffer, 100 ml50 mM Tris (8.0) 5 ml of 1 M stock1 mM EDTA 200 ul of 0.5 M stock
PRODUCTS AND SUPPLIERSProteinase K solution Boehringer Manneheim:1413-783Biotin-16-dUTP Boehringer Manneheim:1093-070Terminal transferase Gibco/BRL:8008SB(TdT)ExtraAvidin Peroxidase SIGMA:E2886AEC Substrate kit Vector:SK-4200Harelco Harris hemotoxylin Baxter:57735-3Crystalmount BioMeda:M02
