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Programmed Cell Death In Situ Detection
Using Terminal deoxynucleotidyl Transferase (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