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Immunohistochemistry

  1. **Optional** Start with antigen retrieval to free the antigen from its crosslink with paraffin. This also helps remove any wrinkles from the section and improves binding.
    1. Put slides on heat block (55°C) until you can see the paraffin starting to melt without the antigen retrieval, the tissue will fall off the slide. The heating will cause the tissue to completely dry out and “stick” to the slide.
  2. De-paraffinize
    1. Xylene, 3min, 3x
  3. Rehydrate
    1. 100% EtOH, 2X, 1min each
    2. 95% EtOH, 2x, 1min each
    3. ddH2O, 1x, 1min
  4. Antigen Retrieval
    1. Heat antigen retrieval buffer (0.01M citric acid pH=6.0 or 0.05% Tween20 in PBS 1X of 10mM Tris, 1mM EDTA pH=8.0 or 0.05% Tween20 in water. The choice of antigen retrieval solution depends on the antibodies you use. Consult manufacturer’s recommendations or published literature.) in microwave for ~3min. *Put the container containing the buffer into another bigger container when heating to prevent overflow into the microwave.
    2. Put slides in the hot buffer (on and off), and heat again for another ~1.5min or until it boils.
    3. Keep boiling on and off for about 20 mins every ~5-7 min to keep slides at boiling temperature for 20 mins.
    4. Let it cool at RT (takes about 20 min).
    5. Pour out buffer and flush with tap water. Put slides in 1X PBS.
  5. Quench: **Silence enzymes which will use H2O2 as a substrate. This willl prevent the interference of these enzymes when H2O2 is added for development.**
    1. 2% H2O2 in PBS (e.g. 13.3 ml "30% H2O2" + 186.7 H2O2 PBS = 200ml "2% H2O2")
    2. Let stand for at least 1 min.
    3. Rinse in 1X PBS, 5 min
  6. Blocking tissue with serum
    1. Vortex serum and centrifuge to homogenize coagulated proteins
    2. Prepare blocking solution (10% serum in 1X PBS): ~100ul for each tissue and additional volume for antibody dilution. **The volume can vary, based on the size of the tissue.
    3. Vacuum the slide to dry it (without drying the actual tissue) and use a blocking pen to draw contour around each piece of tissue on the slide.
    4. Add ~50–100 ul of the blocking solution to each tissue.
    5. Incubate in humidity chamber for ~1hr.
  7. Primary antibody
    1. Add serum to the antibody mix to a final concentration of 2% serum.
    2. Vacuum blocker solution and add desired concentration of primary antibody.
    3. Incubate in humidity chamber for 1-2 hr or overnight at 4°C.
  8. Vacuum the primary antibody and wash slides by dipping several times in 1X PBS. Rinse slides in new 1X PBS, and make sure to keep them wet (Rinse - 5min 3x).
  9. Secondary antibody
    1. Add secondary antibody (1:200 in 20% serum) and incubate in humidity chamber for 1-2hr (RT)
    2. Vacuum 2° and rinse (gently dip and shake) slides in 1X PBS (3x for 5 min each)
  10. Visualization: Follow the instructions on the DAB kit you’re using.
    1. Suction off DAB solution and dip in water or 1X PBS to stop the reaction
    2. Rinse & flush with tap water
    3. ***Counterstain if desired with Hematoxylin***
      1. Hematoxylin ----- 10 dips
      2. Water ------------- Until the solution becomes clear
  11. Dehydrate
    1. 95% EtOH, 2x, 1min each
    2. 100% EtOH, 2x, 1min each
    3. xylene, 3x, 1min
  12. Mount in mounting media; coverslip and dry overnight.
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