Heat Mediated High pH Antigen Retrieving Solution (ab972)

Application notes

Be sure to use heat resistant coplin jars

Procedure: Coplin Jar Method – Dewaxing Method

1. Dry tissue sections at 37°C for 1 hour and then dry slides 10-30 minutes at 60°C.
2. Fill the Coplin Jar with 50 ml of antigen retrieval solution. Fill a second Coplin Jar with 50 ml of deionized water (DI).
3. Place slides in the Coplin Jar.
4. Place both Coplin Jars in the pressure cooker chamber containing 500 ml of deionized water.
5. Place the lid on the chamber and start the heating process
6. When completed, open the chamber and allow solution to cool for 5 minutes.
7. Transfer slides with a forcep into the hot rinse (second Coplin Jar containing the DI water).
8. Continue cooling for 5 minutes.
9. Wash briefly in deionized water and rinse with PBS or TBS.

(See Important Technical Note).

Procedure: Tissue-Tek Staining Dishes - Dewaxing Method

1. Dry tissue sections at 37°C for 1 hour and then dry slides 10-30 minutes at 60°C.
2. Place slides in a slide holder and place in a Tissue-Tek staining dish.
3. Fill the staining dish with 250 ml of antigen retrieval solution. Fill a second staining dish with 250 ml of deionized water (DI).
4. Place both staining dishes in the pressure cooker chamber.
5. Place the lid on the chamber and start the heating process.
6. When completed, open the chamber and allow solution to cool for 5 minutes.
7. Transfer slides into the hot rinse (second Tissue-Tek staining dish containing the DI water).
8. Continue cooling for 5 minutes.
9. Wash briefly in tap water and rinse with PBS.

Procedure: (Non-Dewaxing Method)

1. Deparaffinize tissue and hydrate to water.
2. If necessary, block for endogenous peroxidase.
3. Rinse in DI water.
4. Immerse the slides in antigen retrieval solution.

Heat or boil solution containing the slides for 10-20 minutes. Do not remove the slides from the hot solution. First, cool slides at RT for 10-20 minutes. Then gently rinse slides in cold tap water. After rinsing in tap water, rinse slides in D.I. water. Alternatively, steam tissue sections for 45-60 minutes, or use a pressure cooker for 2-3 minutes


Important Technical Notes:

1. Some antigens may be masked or destroyed, if a hydrogen peroxide block is used prior to primary antibody application. Therefore, we are recommending that the hydrogen peroxide block be performed directly after the primary antibody.
2. There are several heating methods that may be employed. These methods include: microwave oven, pressure cooker, hot water bath or steamer. For microwave use, licensing may be required under U.S. patent No. 5,244,787.
3. If a pressure cooker, heat slides for 2-3 minutes and cool for 10 minutes.