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Mark A. Sanders

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Tracy E. Anderson
Gail Celio

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Chemi Banari
Alex Cramer
Anna Hottinger
Alex Schauer
Ashleigh Skaalen

University of Minnesota
Imaging Center
Rooms 23-35 Snyder Hall
1475 Gortner Avenue.
University of Minnesota,
St. Paul, MN 55108

Hours: 9:00-5:00 weekdays
Closed holidays

Protocol for Microwave-assisted Immuno-labeling

Solutions

• Buffer A - 10 mM Phosphate buffer
  
  
  • 0.01 M phosphate:
  • KH₂PO₄ (monobasic stock, 0.4M) 1.75 ml
  • K₂HPO₄ (dibasic stock, 0.4M) 4.5 ml
  • Q.S. with nH₂O pH 7.2 250.0 ml
  • pH to 7.2

• Buffer B - 10 mM Phosphate buffer + 50 mM NH₄Cl
  
  
  • 0.1 M Phosphate buffer 10.0 ml
  • NH₄Cl (Sigma A-4514, MW 53.5) 0.27 g
  • Q.S. to 100 ml with nH₂O
    

Protocol

1. Rinse sample in Buffer A for 5 min.
   
   
2. Wash 2 times at 1 minute at 0% power, 40 seconds at 100% power, 3 minutes at 0% power in Buffer A.
   
   
3. Wash in Buffer B 1 minute at 0% power, 40 seconds at 100% power, 3 minutes at 0% power to quench free aldehydes formed in glutaraldehyde fixative.
   
   
4. Wash in PBS one time 1 minute at 0% power, 40 seconds at 100% power, 3 minutes at 0% power.
   
   

Immuno-labeling in the Microwave

1. Apply blocking buffer for 2 minutes at 100% power, 2 minutes at 0% power and 2 minutes at 100% power at 37^oC set point.
   
   
2. Remove excess buffer from the slide.
   
   
3. Apply appropriate dilution of primary antibody and incubate at 37^oC for 2 minutes at 100% power, 2 minutes at 0% power, 2 minutes at 100% power and 2 minutes at 0% power.
   
   
4. Wash with PBS three times 40 seconds at 100% power
   
   
5. Apply secondary antibody and incubate at 37^oC for 2 minutes at 100% power, 2 minutes at 0% power, 2 minutes at 100% power and 2 minutes at 0% power.
   
   
6. Wash with PBS three times 40 seconds at 100% power.
   
   
7. Rinse well with distilled water, mount in antifade mounting media and observe.