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

互联网2013-09-05

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<center> 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. </center>

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 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 37^o 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 37^o 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 65^o C 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