Immunohistochemistry Paraffin Protocol

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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:

            a. 70% Ethanol
            b. 95% Ethanol
            c. 100% Ethanol
            d. 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.

IMMUNOFLUORESCENT STAINING

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

            a. Xylene: three times for 5 minutes each
            b. 100% Ethanol: twice for 5 minutes each
            c. 95% Ethanol: 5 minutes
            d. 70% Ethanol: 5 minutes
            e. 50% Ethanol: 5 minutes
            f. 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 use rubber cement.

Tip: Wipe the area around the section with tissue paper before drawing circle. This will help eliminate  the chances of hydrophobic ink’s dispersal over the sections.  

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 permeabilize 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).

Tip: Hydrogen peroxide is a light sensitive chemical, therefore, this step should be (preferably) performed away from direct exposure to light.

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

Tip: Hydrogen peroxide is a light sensitive chemical, therefore, this step should be (preferably) performed away from direct exposure to light.

6. Add primary antibody diluted in 1% animal serum PBS-T and incubate at room temperature for 1-2 hours followed by 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.

Tip: In addition to positive controls, make sure to include the following negative controls in your assay – secondary antibody only (no primary), no primary or secondary antibody.

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

8. Dilute secondary antibody in 1% serum PBS-T and incubate with sections at room temperature for 1-2 hours. Use the  recommended dilution of the antibody as specified on the data  sheet.

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

Optional: Double/Nuclear labeling

a. Double labeling: If using a second primary antibody and  appropriately matched secondary, repeat steps 5-8.

b. Nuclear labeling: After application of all primary antibodies, DNA binding dyes such as DAPI can be applied, After incubation, wash once for 5 minutes with PBS.

10. Tap off excess wash and apply one drop of anti-fade mounting  medium to the slide. Place a coverslip on the tissue  sections. Circle the edges of the coverslip with clear fingernail polish to prevent the cells from drying. Allow nail polish to air dry.

11. Slides may now be examined under a microscope with the  appropriate fluorescent filter sets. Limit the amount of time each slide is exposed to the microscopes light will prolong the signal and prevent photobleading

12. Slides can be stored between -20°C and 4°C in a dark slide box or slide book.

IMMUNOCHROMOGENIC STAINING (ABC METHOD WITH DAB)

1.    Deparaffinize and rehydrate by immersing the slides through the following wells:
            
            a. Xylene: three times for 5 minutes each
            b. 100% Ethanol: twice for 5 minutes each
            c. 95% Ethanol: 5 minutes
            d. 70% Ethanol: 5 minutes
            e. 50% Ethanol: 5 minutes
            f. 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:

            a. 95% Ethanol
            b. 100% Ethanol
            c. 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