Immunocytochemistry Protocol

ICC

Note: Do not let the tissues dry out once they are re‐hydrated.
Use separate tubs for antibodies and negative control slides to avoid contamination.


MATERIALS

  • 4% PFA fixed cell arrays
  • Coverslips
  • Slide racks
  • Staining dishes with lids
  • Plastic slide tray (Baxter Scientific Cat. No. M6304)
  • Orbital shaker
  • Transfer pipettes
  • Deionized water (DI H2O)
  • PBS (Phosphate Buffered Saline)
  • Triton X‐100
  • Hydrogen peroxide (H2O2)
  • Primary antibody
  • Biotinylated secondary antibody, HRP conjugated
  • Bovine Serum Albumin (BSA – for blocking)
  • Streptavidin‐HRP
  • DAB
  • Hematoxylin (optional)
  • Acetic Acid (optional)
  • Glycerol

Permeabilize Membrane (Optional if detecting a membrane protein)

  1. Add one drop of PBS/0.1% Triton X‐100 to each well to permeabilize the cells. Incubate slides for one (1) minute at room temperature.
  2. Remove the liquid and wash the slides twice (2x) in PBS, 5 minutes each on the shaker.
  3. Remove the liquid and place the slides onto a tray.

Blocking

  1. Soak slides in 1.5% H2O2/PBS solution for 15 minutes.
  2. Wash twice (2x) in PBS for 5 minutes each on the shaker.
  3. Incubate with 5% BSA into each well to block for overnight at 4°C in a humid chamber.

Primary Antibody

  1. Dilute the primary antibody to the recommended concentration in 1% BSA diluent.
  2. Remove BSA from the slides.
  3. Add 35μL of primary antibody to each well. Incubate for one (1) hour at room temperature.
  4. Remove the primary antibody solution and wash slides three (3) times in PBS, 5 minutes each on the shaker.

Secondary Antibody and Detection

  1. Dilute the biotinylated secondary antibody to 1:200 in a solution of 1% BSA diluent.
  2. Remove the excess fluid and add one drop secondary antibody solution into each well. Incubate for one (1) hour at room temperature.
  3. Wash in PBS three (3) times 5 minutes each on an orbital shaker. Remove excess fluid.
  4. Add one drop streptavidin‐HRP to each well. Incubate for 30 minutes at room temperature.
  5. Wash three (3) times 5 minutes in PBS on an orbital shaker. Remove excess fluid.
  6. Add DAB solution to each cell well. Once the cells start turning brown (inexperienced technicians may wish to observe this under a microscope) wash twice (2x) in PBS for 5 minutes each time on the shaker.

Optional Counterstain

  1. Dip the slide rack with the slides into a staining dish of hematoxylin for 30 seconds.
  2. Remove and place into an acid bath (200mL DI H2O and one to three drops of acetic acid). Rinse with DI H2O.

Cover Slips

  1. Add several drops of coverslip solution (50% glycerol / DI H2O) to the slide.
  2. Place coverslip on top of the slide.
  3. Store slides at room temperature.