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
  1. 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.