Chromogenic IHC Staining of Formalin-Fixed Paraffin-Embedded Tissue

PREPARATION - PERFUSION AND PARAFFIN EMBEDDING

1. Fix the tissue of interest by immersing it in 10% neutral buffered formalin (4% PFA-PBS) for 4-24 hours at room temperature. Fixation time and temperature depends on tissue type/size. After fixation, wash the tissues thoroughly in PBS.

Tip: Tissues at this stage may be stored in cold PBS until further processing (maximum of 2-3 days). For longer storage, the tissues must be kept in 70% ethanol.

2. Dehydrate by moving tissue through the following solutions twice for 30 minutes each:

  1. 70% Ethanol
  2. 95% Ethanol
  3. 100% Ethanol
  4. Xylene

3. Embed the tissue in molten paraffin. After the paraffin solidifies keep the blocks at 4°C until sectioning.

4. Use a microtome to cut the embedded tissue into 4-6 µm thick sections and float them in a 50°C water bath containing distilled water.

5. Mount sections onto gelatin or poly-L-lysine coated slides and allow them to dry overnight. Slides can be safely stored at room temperature until ready for staining.

IMMUNOCHROMOGENIC STAINING (ABC METHOD WITH DAB)

1. Deparaffinize and rehydrate by immersing the slides through the following wells:

  1. Xylene: three times for 5 minutes each
  2. 100% Ethanol: twice for 5 minutes each
  3. 95% Ethanol: 5 minutes
  4. 70% Ethanol: 5 minutes
  5. 50% Ethanol: 5 minutes
  6. Distilled water: 5 minutes. Do not let the tissue dry from this point on.

Tip: Before moving to alcohol grades step, make sure to completely deparaffinize the sections. If the sections still have traces of wax, an additional immersion of 5 minutes in Xylene may be employed.

2. Draw a circle on the slide around the tissue with a hydrophobic barrier pen or with rubber cement.

3. For antigen retrieval using microwave, bring the slides to a boil in 10 mM sodium citrate buffer (pH 6.0) and then maintain at a sub-boiling temperature for 10 minutes. Then, let it cool on bench-top for about 30 minutes and wash the sections by immersing them in distilled water for 5 minutes.

4. To block endogenous peroxidase activity, quench the tissue sections with 3.0% hydrogen peroxide in methanol for at least 15 minutes. Afterwards, wash the sections by immersing them in distilled water for 5 minutes.

5. To permeablize the tissue/cells, wash the sections twice for 10 minutes with 1% animal serum in PBS with 0.4% Triton X-100 (PBS-T). The species of the animal serum is dependent on the host of your secondary antibody. (e.g. when using a goat anti-mouse secondary, use goat serum).

6. Block any non-specific binding by incubating the tissue sections with 5% animal serum in PBS-T for 30 minutes at room temperature.

7. Add primary antibody diluted in 1% animal serum PBS-T and incubate at room temperature for 1-2 hours. Then store overnight at 4°C in humidified chamber. Use the recommended dilution of the antibody specified on the datasheet. If not specified, the typical starting dilution can be 2-5 µg/ml.

8. Wash sections twice with 1% serum PBS-T for 10 minutes each.

9. Add a biotinylated secondary antibody and incubate at room temperature for 1 hour. Use the recommended dilution of the antibody specified on the datasheet.
10. Wash sections twice with 1% serum PBS-T for 10 minutes each.

11. Add ABC-HRP reagent and incubate at room temperature for 1 hour. Follow manufacturer’s guidelines for reagent preparation.

12. Wash sections twice in PBS for 10 minutes each.

13. Important: DAB is a carcinogen! Always wear gloves and work in a fume hood when working with DAB. Deactivate and clean work area after use according to manufacturer’s instructions. Prepare a working solution of DAB and apply to tissue sections. Monitor the reaction as the chromogenic reaction turns the epitope sites brown (time of color development may vary from few seconds to 10 minutes). Proceed to the next step when the intensity of the signal is appropriate for imaging.

14. Wash the sections twice in distilled water for 2 minutes each.

15. To counterstain nuclei, use Hematoxylin according to the manufacturer’s instructions. Note: If you are using an aqueous chromogen instead of DAB (i.e. AEC, Fast Red, etc.), skip the following dehydration step and mount in aqueous media instead of organic mounting media.

16. Dehydrate tissue sections by moving slides through the following solutions twice for 2 minutes each:

  1. 95% Ethanol
  2. 100% Ethanol
  3. Xylene

17. Add mounting media to slides and top with coverslips. The DAB reaction is permanent and stable and can be analyzed under a brightfield microscope at any time.

More Resources:

Immunohistochemistry Paraffin (IHC-P) Troubleshooting