IHC antigen retrieval protocol

Description:

 Formalin or other aldehyde fixation forms protein cross-links that mask the antigenic sites in tissue specimens, thereby giving weak or false negative staining for immunohistochemical detection of certain proteins. The citrate based solution is designed to break the protein cross-links, therefore unmask the antigens and epitopes in formalin-fixed and paraffin embedded tissue sections, thus enhancing staining intensity of antibodies.

 

Solutions and Reagents for heat-induced epitope retrieval(HIER):

1. Sodium Citrate Buffer (10mM Sodium Citrate, 0.05% Tween 20, pH 6.0):

      Tri-sodium citrate (dihydrate) --------- 2.94 g

      Distilled water --------------------------- 1000 ml

      Mix to dissolve. Adjust pH to 6.0 with 1N HCl and then add 0.5 ml of Tween 20 and mix well. Store this solution at room temperature for 3 months or at 4 C for longer storage.

      Note: this buffer is commonly used and works perfectly with many antibodies. It gives very nice intense staining with very low background.

 

2. Citrate Buffer (10mM Citric Acid, 0.05% Tween 20, pH 6.0):

      Citric acid (anhydrous)  --------------- 1.92 g

      Distilled water -------------------------- 1000 ml

      Mix to dissolve. Adjust pH to 6.0 with 1N NaOH and then add 0.5 ml of Tween 20 and mix well. Store this solution at room temperature for 3 months or at 4 C for longer storage.

 

3. Citrate-EDTA Buffer (10mM Citric Acid, 2mM EDTA, 0.05% Tween 20, pH 6.2):

      Citric acid (anhydrous)  ----------------- 1.92 g

      EDTA (Sigma, Cat# E-5134) ------------ 0.74 g

      Distilled water --------------------------- 1000 ml

     Mix to dissolve. Adjust pH to 6.2 and then add 0.5 ml of Tween 20 and mix well. Store this solution at room temperature for 3 months or at 4 C for longer storage.

 

4. EDTA Buffer (1mM EDTA, 0.05% Tween 20, pH 8.0):

      EDTA (Sigma, Cat# E-5134) ----------- 0.37 g

      Distilled water -------------------------- 1000 ml

      Mix to dissolve. Adjust pH to 8.0 using 1N NaOH. Then add 0.5 ml of Tween 20 and mix well. Store this solution at room temperature for 3 months or at 4 C for longer storage.

      Note: This buffer works excellent for many antibodies, but it often gives high background staining (maybe due to endogenous biotin revealed after this pretreatment). So primary antibody can often be highly diluted. It is very useful for low affinity antibodies or when tissue antigens are not intense.

 

Methods for HIER: pressure cooker

1) Add the appropriate antigen retrieval buffer to the pressure cooker. Place the pressure cooker on the hotplate and turn it on full power. Do not secure the lid of the pressure cooker at this point, simply rest it on top. While waiting for the pressure cooker to come to a boil, de-paraffinize and rehydrate the sections.

2) Once boiling, transfer the slides from the tap water to the pressure cooker.

Use care with hot solution – use forceps.

3) Secure the pressure cooker lid as per the manufacturer’s instructions.

4) As soon as the cooker has reached full pressure, time 3 min.

5) When 3 min have elapsed, turn off the hotplate and place the pressure cooker in an empty sink.

6) Activate the pressure release valve and run cold water over the cooker. Once de-pressurized, open the lid and run cold water into the cooker for 10 min.

This is to allow the slides to cool enough so they may be handled, and to allow the antigenic site to re-form after being exposed to high temperature.

7) Continue with the immunohistochemical staining protocol.

 

Methods for HIER: microwave

1) Deparaffinize and rehydrate the sections.

Use a sufficient volume of antigen retrieval solution to cover the slides by at least a few centimeters.

2) Add the appropriate antigen retrieval buffer to the microwaveable vessel.

Use a non-sealed vessel to allow for evaporation during the boil. Be sure to monitor for evaporation and watch out for boiling over during the procedure and do not allow the slides to dry out.

3) Place the slides in the microwaveable vessel. Place the vessel inside the microwave. If using a domestic microwave, set to full power and wait until the solution comes to the boil. Boil for 20 min from this point. If using a scientific microwave, program so that antigens are retrieved for 20 min once the temperature has reached 98°C.

 

Use a non-sealed vessel to allow for evaporation during the boil. Be sure to monitor for evaporation and watch out for boiling over during the procedure and do not allow the slides to dry out.

4) When 20 min has elapsed, remove the vessel and run cold tap water into it for 10 min. Use care with hot solution.

This allows the slides to cool enough so they may be handled, and allows the antigenic site to re-form after being exposed to high temperature.

5) Continue with the immunohistochemical staining protocol.

 

 

IHC Procedure:

1. Deparaffinize sections in 2 changes of xylene, 5 minutes each.

2. Hydrate in 2 changes of 100% ethanol for 3 minutes each, 95% and 80% ethanol for 1 minute each. Then rinse in distilled water.

3. Pre-heat steamer or water bath with staining dish containing Sodium Citrate Buffer or Citrate Buffer until temperature reaches 95-100 °C.

4. Immerse slides in the staining dish. Place the lid loosely on the staining dish and incubate for 20-40 minutes (optimal incubation time should be determined by user).

5. Turn off steamer or water bath and remove the staining dish to room temperature and allow the slides to cool for 20 minutes.

6. Rinse sections in PBST for 3x5 min.

7. Block sections with for 30 minutes.

8. Perform avidin/biotin blocking if necessary.

9. Incubate sections with primary antibody at appropriate dilution in primary antibody dilution buffer for 1 hour at room temperature or overnight at 4 °C.

10. Rinse sections with PBST for 3x5 min.

11. Block sections with peroxidase blocking solution for 10 minutes.

12. Rinse with PBST for 3x5 min.

13. Proceed to standard immunohistochemistry protocol.

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