Immunocytochemistry (ICC) Protocol
Reagents Required
Product |
Phosphate Buffered Saline (PBS) |
Triton X-100 |
Hydrogen Peroxide (H2O2) |
Bovine Serum Albumin (BSA) |
Biotinylated Secondary Antibody, 1:500 |
Streptavidin Peroxidase Conjugated, 1:500 |
3,3'-Diaminobenzidine (DAB) Substrate |
Hematoxylin (Optional) and Acetic Acid (Optional) for counterstain |
UltraPure Sterile Water |
Coverslip Solution (50% glycerol/UltraPure water) |
Procedure
Note: This procedure is optional if detecting a membrane protein.
- Add one drop of PBS/0.1% Triton X‐100 to each well to permeabilize the cells and incubate slides for one 1 minute at room temperature.
- Remove the liquid and wash the slides twice in PBS, 5 minutes each on the shaker.
- Remove the liquid and place the slides onto a tray.
- Soak slides in 1.5% H2O2/PBS solution for 15 minutes.
- Wash twice 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 35 µL of primary antibody to each well. Incubate for one 1 hour at room temperature.
- Remove the primary antibody solution and wash slides 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 3 times, 5 minutes each on an orbital shaker. Remove excess fluid.
- Add one drop streptavidin peroxidase to each well. Incubate for 30 minutes at room temperature.
- Wash 3 times, 5 minutes in PBS on an orbital shaker. Remove excess fluid.
- Add DAB substrate to each cell well. Once the cells start turning brown wash 2 times in PBS for 5 minutes each on the shaker.
Note: Inexperienced technicians may wish to observe cells turning brown under a microscope. - (Optional step for counterstain) Dip the slide rack with the slides into a staining dish of hematoxylin for 30 seconds. Remove and place into an acid bath (200 mL UltraPure water and 1–3 drops of acetic acid). Rinse with UltraPure water.
- Add several drops of coverslip solution.
- Place the coverslip on top of the slide.
- Store slides at room temperature.