组织在制作过程中，由于化学试剂的作用封闭了抗原，又由于热的作用致使部分抗原的肽链发生扭曲，致使在免疫组化的染色过程中不能将其显示出来，为了解决上述的问题，利用化学试剂和热的作用将这些抗原重新暴露出来或修正过来的过程称为抗原修复。柠檬酸盐、EDTA或Tris等缓冲液在热的条件下可以使被福尔马林屏蔽的抗原重新暴露出来，同时又不会对抗原表位造成破坏，从而提高抗原的检出率，降低背景染色，提高诊断的准确率。
Tris抗原修复液 (10×, pH 9.5)由Tris、微量Tween20等组成，可以有效去除醛类固定试剂导致的蛋白之间的交联，充分暴露石蜡切片等样品中的抗原表位，可以用于石蜡切片、冰冻切片等样品使用多聚甲醛、甲醛或其它醛类试剂固定后的抗原修复；抗原修复可以提高石蜡切片的免疫染色效果，亦可以不同程度的提高冰冻切片的染色效果；当冰冻切片免疫染色效果不理想时，可考虑进行抗原修复，按照每个片子需要10ml抗原修复液 (1×)计算，100ml抗原修复液 (10×)可以用于100个样本的抗原修复。该试剂仅用于科研领域，不适用于临床诊断或其他用途。
自备材料：
1、系列乙醇
2、双蒸水或去离子水
3、加热设备
4、免疫染色洗涤液
操作步骤(仅供参考)：
(一)石蜡切片
1、脱蜡至水
①二甲苯或脱蜡透明液脱蜡3次，每次3-5min。
②无水乙醇脱水2次，每次3-5min。
③95%的乙醇，3-5min。
④90%的乙醇，3-5min。
⑤80%的乙醇，3-5min。
⑥70%的乙醇，3-5min。
⑦蒸馏水冲洗2次，每次3-5min。
2、抗原修复
①用去离子水或双蒸水稀释Tris抗原修复液 (10×, pH 9.5)至1×。
②将切片浸泡在Tris抗原修复液 (1×)中，95℃或沸水加热约10-30min。
③抗原修复液 (1×)使用前需预热到95-100℃，如果使用微波炉加热，避免暴沸和过多的水分蒸发，随后大约在20-30min内冷却至室温。
3、免疫染色洗涤液洗涤1-2次，每次3-5min。
4、封闭等后续的免疫染色步骤。
(二)冰冻切片
1、用去离子水或双蒸水稀释Tris抗原修复液 (10×, pH 9.5)至1×。
2、免疫染色洗涤液洗涤切片5min。
3、将切片浸泡在抗原修复液 (1×)中，95℃或沸水加热约10-30min。
4、抗原修复液 (1×)使用前预热至95-100℃，如果使用微波炉加热，避免暴沸和过多的水分蒸发，随后大约在20-30min内冷却至室温。
5、免疫染色洗涤液洗涤1-2次，每次3-5min。
6、进行封闭等后续的免疫染色步骤。
(三)其它样品
其它样品参考石蜡切片或冰冻切片进行操作。
注意事项：
1、浸泡在抗原修复液 (1×)中，最佳的加热时间需根据不同的样品和目的蛋白自行摸索。
2、为了您的安全和健康，请穿实验服并戴一次性手套操作。
3、试剂开封后请尽快使用，以防影响后续实验效果。
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.
ZJ26F0232088
危险品化学品经营许可证（带存储）