Indirect Immunofluorescence

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>

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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>

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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>
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