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.
ALL STEPS ARE PERFORMED AT ROOM TEMPERATURE
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.
THE REST OF THE STEPS ARE PERFORMED IN THE DARK-EITHER PLACE THE SLIDE IN A DRAWER OR COVER IT WITH FOIL
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.
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