Programmed Cell Death In Situ Detection Using Terminal deoxynucleotidyl Transfer(TdT)

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 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 37oC 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 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.

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