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

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        1371

         

        <center> <p> <font><font><font color="#090809"><b>Programmed Cell Death In Situ Detection</b><br /> <b>Using Terminal deoxynucleotidyl Transferase (TdT)</b> </font> </font> </font></p> <p> <b><font color="#090809">Terminal deoxynucleotidyl Transferase-mediated dUTP nick end labeling (TUNEL) is an <i>in situ</i> 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.</font> </b></p> </center>

        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 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 37o 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 37o 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 65o 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

         

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