During tissue preparation, antigens are masked by the effects of chemical reagents, and the peptide chains of some antigens become distorted due to heating. As a result, these antigens cannot be visualized during immunohistochemical staining. To address these issues, the process of re-exposing or restoring these antigens using chemical reagents and heat is called antigen retrieval. Buffers such as citrate, EDTA, or Tris can re-expose antigens masked by formalin under heating conditions without damaging antigenic epitopes, thereby improving antigen detection rates, reducing background staining, and enhancing diagnostic accuracy.
Tris Antigen Retrieval Solution (10×, pH 9.5) is composed of Tris, a trace amount of Tween 20, and other ingredients. It can effectively eliminate protein cross-linking caused by aldehyde-based fixatives and fully expose antigenic epitopes in samples such as paraffin sections and frozen sections. This solution is suitable for antigen retrieval of samples (including paraffin sections and frozen sections) fixed with paraformaldehyde, formaldehyde, or other aldehyde-based reagents. Antigen retrieval can improve the immunostaining effect of paraffin sections and enhance the staining effect of frozen sections to varying degrees. When the immunostaining effect of frozen sections is unsatisfactory, antigen retrieval is recommended. Based on a dosage of 10 ml of Tris Antigen Retrieval Solution (1×) per slide, 100 ml of the 10× concentrated solution can be used for antigen retrieval of 100 samples. This reagent is intended for research purposes only and not for clinical diagnosis or other applications.
Materials to Be Prepared by Users
1. Graded ethanol solutions
2. Double-distilled water or deionized water
3. Heating equipment
4. Immunostaining wash buffer
Operating Procedures (For Reference Only)
(1) Paraffin Sections
1. Deparaffinization and Rehydration
① Deparaffinize sections with xylene or deparaffinization clearing solution for 3 times, 3-5 minutes each time.
② Dehydrate with anhydrous ethanol for 2 times, 3-5 minutes each time.
③ Treat with 95% ethanol for 3-5 minutes.
④ Treat with 90% ethanol for 3-5 minutes.
⑤ Treat with 80% ethanol for 3-5 minutes.
⑥ Treat with 70% ethanol for 3-5 minutes.
⑦ Rinse with distilled water for 2 times, 3-5 minutes each time.
2. Antigen Retrieval
① Dilute Tris Antigen Retrieval Solution (10×, pH 9.5) to 1× working concentration with deionized water or double-distilled water.
② Immerse sections in the 1× Tris Antigen Retrieval Solution, and heat at 95 °C or boiling temperature for approximately 10-30 minutes.
③ Preheat the 1× Tris Antigen Retrieval Solution to 95-100 °C before use. If heating with a microwave oven, avoid boiling and excessive water evaporation. Then allow the solution to cool down to room temperature naturally within approximately 20-30 minutes.
3. Wash sections with immunostaining wash buffer for 1-2 times, 3-5 minutes each time.
4. Proceed with subsequent immunostaining steps, such as blocking.
(2) Frozen Sections
1. Dilute Tris Antigen Retrieval Solution (10×, pH 9.5) to 1× working concentration with deionized water or double-distilled water.
2. Wash sections with immunostaining wash buffer for 5 minutes.
3. Immerse sections in the 1× Tris Antigen Retrieval Solution, and heat at 95 °C or boiling temperature for approximately 10-30 minutes.
4. Preheat the 1× Tris Antigen Retrieval Solution to 95-100 °C before use. If heating with a microwave oven, avoid boiling and excessive water evaporation. Then allow the solution to cool down to room temperature naturally within approximately 20-30 minutes.
5. Wash sections with immunostaining wash buffer for 1-2 times, 3-5 minutes each time.
6. Proceed with subsequent immunostaining steps, such as blocking.
(3) Other Samples
Follow the procedures for paraffin sections or frozen sections.
Precautions
1. The optimal heating time for sections immersed in the 1× Tris Antigen Retrieval Solution should be determined empirically according to different samples and target proteins.
2. For your safety and health, wear a lab coat and disposable gloves during operation.
3. Use the reagent as soon as possible after opening to avoid compromising subsequent experimental results.
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