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Immunofluorescent staining of adherent differentiated ES cell embryoid bodies

1. Differentiate ES cells as per STD protocol except after at least 4 days in suspension, transfer to cover slides coated with 0.1% gelatin.
   
   
2. After 10 days total, cells can be stained with cardiac markers: after 15-25 days cells can be stained for smooth muscle markers.
   
   
3. To stain cells, remove media from slides and wash cells 2X with PBS +/+.
   
   
4. Fix cells with either 100% EtOH or 100% MeOH for 10 min at -200C.
   -EtOH is more gentle for Abs that dont work as well for immunofluorescent staining
   
   
5. Air dry cells for 5-10 min and then rehydrate with PBS+/+ 2X at RT for 5 min.
   
   
6. Pre-block and permeablize cells with PBS + 10% goat serum (or rabbit serum if secondary Ab is from rabbit) + 0.1% NP-40, RT for 20 min.
   
   
7. Remove pre-block and add primary Ab at desired concentration ( e.g. use sm-actin at 5 ug/ml) in PBS + 10% goat (or rabbit) serum. Incubate ON at RT in a humid chamber.
   
   
8. Next morning, remove primary Ab and wash 6X 30 min each with PBS +/+.
   
   
9. Add secondary Ab at desired concentration in PBS + serum and incubate at RT for 2hrs. with gentle mixing.
   
   
10. After 2 hrs., wash 4X 30 min each with PBS.
    
    
11. OPTIONAL-counter stain with DAPI for 1min in PBS and wash 2X 15min each with PBS.
    
    
12. Mount with Vectaseal and a coverslip--can post-fix with 3.7% formaldehyde for 15 min at RT and wash an additional 2X 10 min. with PBS-------then view under fluorescent microscope!!!

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