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Posts Tagged ‘AT1’

Visualizing AT1 Receptors Using Immunohistochemistry

Here’s a condensed version of the modified protocol we assembled to visualize the AT1 receptors on the brain tissues of hampsters….

  1. Wash the slides 3x in 0.1M PBS for 5 min
  2. Wash in .075% H2O2 for 30 min in .1M PBS
  3. Wash the slides 3x in 0.1M PBS for 5 min
  4. Incubate in the Primary Antibody (1:500) in a buffer solution containing 10% goat serum, 0.1% triton X-100, 0.05% tween-20 in 0.1M PBS for 2 hours at room temp then for another 48 hours at 4°C.
  5. Wash the slides 3x in 0.1M PBS for 5 min
  6. Incubate in the Secondary Antibody (1:250) for 50 min at room temp in the buffer solution
  7. Wash the slides 3x in 0.1M PBS for 5 min
  8. Incubate in Streptavidin-Horseradish Peroxidase (1:150) in a TNB blocking buffer solution for 30 min.
  9. Wash 3x in TNT wash for 5 min
  10. Amplification for 5 min in DMSO (1:50) in amplification diluent.
  11. Wash the slides 3x in 0.1M PBS for 5 min
  12. Incubate in ABC kit for 30 min
  13. Wash the slides 3x in 0.1M PBS for 5 min
  14. Incubate in DAB (6000µl 0.1M PBS, 2.4mg DAB, 15µl) for 2-10 min until desired coloration is obtained