All steps done at room temp; can be done at 4C if necessary but must be left overnight (for primary antibody step, e.g)


  1. Wash sections 2x with 1xTBS, 10 minutes each wash
  2. Add enough 0.1% Triton X in TBS to cover section, leave 10 minutes
  3. Wash 2x with 1x TBS, 10 minutes each
  4. Block with 5% BSA in TBS for 60 minutes
  5. Add primary antibody in TBS + 0.8% BSA for 120 minutes
  6. Wash 4x with TBS, 10 minutes each
  7. Block for second antibody in normal serum (5% in TBS) of the host animal 15 minutes (e.g., if secondary antibody is made in goat, use goat serum; if donkey, use donkey serum, etc.)
  8. Add secondary antibody (labeled e.g. with rhodamine) for 60 minutes
  9. Wash 4 x in 1x TBS 10 minutes each
  10. Cover slip with medium for non-dehydrated sections.