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Immunocytochemistry (ICC) Protocol

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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
  • Soak slides in 1.5% H2O2/PBS solution for 15 minutes.
  • Wash twice (2x) in PBS for 5 minutes each on the shaker.
  • Incubate with 5% BSA into each well to block for overnight at 4°C in a humid chamber.
  • Soak slides in 1.5% H2O2 /PBS solution for 15 minutes.
  • Wash twice (2x) in PBS for 5 minutes each on the shaker.
  • Incubate with 5% BSA into each well to block for overnight at 4°C in a humid chamber.
  • Dilute the primary antibody to the recommended concentration in 1% BSA diluent.
  • Remove BSA from the slides.
  • Add 35μL of primary antibody to each well. Incubate for one (1) hour at room temperature.
  • Remove the primary antibody solution and wash slides three (3) times in PBS, 5 minutes each on
    the shaker.
  • Dilute the biotinylated secondary antibody to 1:200 in a solution of 1% BSA diluent.
  • Remove the excess fluid and add one drop secondary antibody solution into each well. Incubate
    for one (1) hour at room temperature.
  • Wash in PBS three (3) times 5 minutes each on an orbital shaker. Remove excess fluid.
  • Add one drop streptavidin‐HRP to each well. Incubate for 30 minutes at room temperature.
  • Wash three (3) times 5 minutes in PBS on an orbital shaker. Remove excess fluid.
  • Add DAB solution to each cell well. Once the cells start turning brown (inexperienced
    technicians may wish to obs
  • Dip the slide rack with the slides into a staining dish of hematoxylin for 30 seconds.
  • Remove and place into an acid bath (200mL DI H2O and one to three drops of acetic acid). Rinse
    with DI H2O.
  • Add several drops of coverslip solution (50% glycerol / DI H2O) to the slide.
  • Place coverslip on top of the slide.
  • Store slides at room temperature.