IHC Counterstaining Protocol

DAPI Counterstaining:

  • 1. Wash and equilibrate the sections with PBS for 5 min*3.
  • 2. Dilute the DAPI solution to an appropriate concentration of working solution as the manual instruction.
  • 3. Drop appropriate amount of DAPI working solution to the slides as the manual instruction (Normally 300-400µl) and make sure the tissue is all covered by the solution.
  • 4. Incubate at room temperature for 5 min.
  • 5. Wash in PBS bath for 5 min*3.
  • 6. Mount the sections with mounting medium and coverslip.

To make this step simple, mounting medium (DAPI-contained) is recommended.

Haematoxylin Eosin (H&E) Counterstaining:

  • 1. Counterstain in eosin-phloxine solution for 30 seconds to 1 minute.
  • 2. Dehydrate through 95% alcohol, 2 changes of absolute alcohol, 5 minutes each.
  • 3. Clear in 2 changes of xylene, 5 minutes each.
  • 4. Mount with xylene based mounting medium.