IHC mouse antibody protocol

E-Cadherin Staining

Solution and kits

Item Item # Manufacturer
Vector MOM Kit BMK-2202 Vector Laboratories, Inc.
DAB Chromogen K3465 Dako
Elite ABC Kit PK6100 Vector Laboratories, Inc.
Biotin Blocking System X0590 Vector Laboratories, Inc.
Mayer's Hematoxylin Soln MHS32-1L Sigma

Procedure

  1. Heat slides in an oven at 60°C for 45 minutes. (usually do a day before)
  2. Deparaffinize/hydrate using the following series of washes:

    Xylenes 3X 5 min
    100% EtOH 2X 2 min
    95% EtOH 2X 2 min
    80% EtOH 1X 2 min
    70% EtOH 1X 2 min
    dH2O 2X 3 min + shaker
    PBS 1X 5 min + shaker




  3. Microwave slide in boiling 10mM Sodium Citrate Buffer. Fill chamber to brim with buffer. Microwave 10 minutes on high power in buffer and allow 20 minutes to cool down at RT.
  4. Prepare MOM + Avidin block solution: 100 ul/2 drops of MOM blocking reagent + 0.5 ml/10 drops Avidin per 2.5ml PBS
  5. Prepare MOM + Biotin solution: 200 ul of MOM protein concentrate + 0.5 ml/10drops Biotin per 2.5 ml PBS
  6. Rinse as the following:

    dH2O 3X 2 min
    3% dH2O2 1X 10 min-never let slide dry
    dH2O 1X 2 min
    Outline with PAP pen
    dH2O 2X 2 min
    PBS 1X 5 min
    MOM + Avidin 1X 1hr
    PBS 2X 2 min
    MOM + Biotin 1X 5 min


  7. Dilute primary antibody in MOM + Biotin solution. Incubate primary antibody for 30 min. Start to prepare ABC Elite reagent mix because it has to sit at RT for 30 min before use.
  8. Prepare ABC elite reagent mix: 2 drops of each A + B per 5 ml PBS
  9. Wash slide with PBS twice and 2 min per wash.
  10. Prepare secondary antibody in MOM diluent (200 ul MOM protein concentrate + 2.5 ml PBS), 10 ul of stock MOM kit secondary antibody in 2.5 ml MOM diluent.
  11. Incubate with secondary antibody for 10 min.
  12. Wash with PBS three times with five minutes each.
  13. Incubate with ABC elite reagent for 30 min.
  14. Wash with PBS three times with five minutes each.
  15. Prepare DAB mix: 1 ml of DAB buffer + 1 drops DAB, Vortex to mix.
  16. Apply DAB until the section turns brown. (Do not incubate for more than 5 min)
  17. Stop the reaction by putting into dH2O.
  18. Rinse as the following:

    Hematoxylin (Sigma) 1X 30 sec
    dH2O 2X 5 min
    95% EtOH 2X 10 sec
    100% EtOH 2X 10 sec
    Xylenes 2X 10 sec


  19. Add non aqueous mounting medium (permamount) and coverslip.
  20. Let sit on level surface for dry.