HORSERADISH PEROXIDASE (HRP) - DAB ON CELLS IN CULTURE

• Cells incubated for 0, 5 or 30 min HRP
   
• Washed with medium without proteins
   
• Washed in 0.14 M cacodylate buffer
   
• Fixed for 30 min at room temperature with 2 % glutaraldehyde in 0.1 M cacodylate buffer pH 7.4
   
• Rinsed in 0.1 M cacodylate buffer
   
• Wash 2 x 50 mM tris buffer pH 6.0
   
• Incubation for 15 min: 50 mM Tris/HCl pH 6.0 with 2 mg/ml DAB and 25 mM merthiolate (thimerosal) (i.e. 101 mg -> 10 ml DAB) (solution is filtered on a 0.2 µm acrodisk; kept in dark)
   
• Add to that solution for 30 min: H₂O₂ (final concentration 0.01 % from a 1 % solution)
   
• Rinse 5 x 5 min with Tris buffer
   
• Rinse in water
   
• Post-fix for 1 h at 4°C with 1 % OsO₄ with 2% K₄Fe(CN)₆
   
• Agar Pellet
   
• Dehydration and Spurr embedding