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        Indirect Immunofluorescence

        互联网

        2398

        Reagents:
         NGS-Normal Goat Serum
         PBST-0.5% Tween-20/CMF-PBS
         CMF-PBS
         1°Ab-mouse MF20 (anti-myosin monoclonal)
         1°Ab-rabbit myoD (anti-myoD polyclonal)
         2°Ab-goat anti-mouse IgG-FITC
         2°Ab-goat anti-rabbit IgG-FITC

        Procedure:

        You will be staining one of your transfected chamberslides with anti-myoD.

        You will be staining the other transfected chamberslide and the untransfected one with anti-myosin.

        <center> <p>  </p> <hr /> <font><font><font>ALL STEPS ARE PERFORMED AT ROOM TEMPERATURE</font> </font> </font></center>

        1.  Block 30 minutes in 1% NGS/CMF-PBS--use 500 ul/well.

        2.  Dilute the primary antibodies in 1% NGS/CMF-PBS.
         Make 4 mls MF20 diluted 1:5
         Make 1.5 mls myoD diluted 1:100

        3.  Bind the primary antibodies 1.5 hours at room temperature in 1% NGS/CMF-PBS--use 300 ul/well.

        4.  Wash 3X with PBST, 5 minutes each wash-use ~1 ml/well.

        <center> <p>  </p> <hr /> <font><font>THE REST OF THE STEPS ARE PERFORMED IN THE DARK-EITHER PLACE THE SLIDE IN A DRAWER OR COVER IT WITH FOIL</font> </font></center>

        5.  Dilute secondary antibodies in 1% NGS/CMF-PBS.
         Make 4 mls anti-mouse FTIC diluted 1:150
         Make 1.5 mls anti-rabbit FITC diluted 1:200

        6.  Bind secondary antibodies 45 minutes--use 300 ul/well.

        7.  Wash 3X with PBST, 5 minutes each wash-use ~1 ml/well.

        8.  Wash 1X with CMF-PBS, 5 minutes-use ~1 ml/well.

        9.  Mount with 20 ul of Vectashield with DAPI/well, add coverslip.  Drain excess Vectashield by holding slide on its side on a piece of filter paper.

        10.  Store at 4°C in a slide box or view immediately.

        <center> <p>  </p> </center>
        上一篇:免疫荧光操作(共聚焦显微镜)-Immunofluorescence Protocol for Confocal Microscopy   下一篇:酵母免疫荧光染色--Immunofluorescent Staining of Yeast:SDS Permeabilization Metho
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