UCSF

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

  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.
  2. Prepare MOM + Avidin block solution: 100 ul/2 drops of MOM blocking reagent + 0.5 ml/10 drops Avidin per 2.5ml PBS
  3. Prepare MOM + Biotin solution: 200 ul of MOM protein concentrate + 0.5 ml/10drops Biotin per 2.5 ml PBS
  4. Rinse as the following:

dH2O

3X 2 min

3% H2O2

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 1 hr

PBS

2X 2 min

MOM + Biotin

1X 5 min

  1. 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.
  2. Prepare ABC elite reagent mix: 2 drops of each A + B per 5 ml PBS
  3. Wash slide with PBS twice and 2 min per wash.
  4. 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.
  5. Incubate with secondary antibody for 10 min.
  6. Wash with PBS three times with five minutes each.
  7. Incubate with ABC elite reagent for 30 min.
  8. Wash with PBS three times with five minutes each.
  9. Prepare DAB mix: 1 ml of DAB buffer + 1 drops DAB, Vortex to mix.
  10. Apply DAB until the section turns brown. (Do not incubate for more than 5 min)
  11. Stop the reaction by putting into dH2O.
  12. 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

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