IHC rabbit antibody protocol

B-Cadherin Immunohistochemical Staining

Solution and reagents

Sodium Citrate Buffer (10mM Sodium Citrate, 0.05% Tween 20, pH 6.0)

Item Quantity
Tri-sodium citrate (dehydrate) Sigma #S-4641 2.94g
Distilled water

1000ml

Mix to dissolve. Adjust pH to 6.0 with HCL or NaOH and then add 0.5ml of Tween 20 and mix. Store this solution at room temperature for 3 months or at 4°C for longer storage.

Item Item # Manufacturer
Vector MOM Kit BMK-2202 Vector Laboratories Inc.
Avidin/Biotin X0590 Dako
Goat-anti-M IgG Biotin Conjugated 81-6540 Zymed
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.

Staining procedure

  1. Wipe excess fluid off slide with Kimwipe and outline the specimen area with PAP pen.
  2. Rinse sections in PBS 2X 2 minutes.
  3. Incubate sections for 20 minutes with 10% goat serum (Diluted normal blocking serum from the species in which the secondary antibody is made).
  4. Perform avidin/biotin blocking. Add 3-5 drops of Avidin, RT, 10 minute pipet tip box.
  5. Rinse sections with 1X PBS with shaking. Repeat with Biotin.
  6. Incubate sections for 1 hour at RT with primary antibody mouse anti-B-catenin (1:200) dilution in PBS.
  7. Rinse sections with PBS 2X 3 minutes
  8. Quench endogenous peroxidase activity by incubating sections 10 minutes in 3% H2O
  9. Rinse sections with PBS 2X 3minutes.
  10. Incubate sections for 30 minutes with secondary antibody goat anti-mouse biotin conjugated (1:500 dilution in PBS).
  11. Rinse sections with PBS 5 minutes.
  12. Incubate sections for 30 minutes with VECTASTAIN elite ABC Reagent: Ad exactly 2 drops of Reagent A (gray label) to 5ml of PBS. Then add exactly 2 drops of Reagent B to the mixture. Mix immediately and allow to stand for 30 minutes before use.
  13. Wash slides for 5 minutes in PBS.
  14. Incubate sections in DAB for maximum time of 5 minutes (1ml DAB buffer + 1 drop DAB spin mixture.) Stop reaction by placing slide in water.
  15. Filter the Mayer’s hematoxylin and immerse slide in Mayer’s hematoxylin for 1 minute.
  16. Dip in tap water.
  17. Incubate the slide into PBS for 2 minutes.
  18. Dehydrate slides in graded alcohols: 50%, 70%, 95%, 100% for 20s each.
  19. Place in Xylene for 2 minutes.
  20. Coverslip slide with clean mount mounting medium.