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。
在热诱导抗原修复法出现之前，胰蛋白酶、蛋白酶K、胃蛋白酶等酶类的诱导的表位修复是最常用的方法。该胰蛋白酶抗原修复液主要由胰蛋白酶溶解于氯化钙溶液中，pH值约为7.8，能暴露石蜡切片等样品中的抗原表位，从而大大改善免疫染色效果，通常石蜡切片都需进行抗原修复处理，而冰冻切片可以不进行抗原修复处理，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10 ml抗原修复液 (1×)计算，100 ml抗原修复液可以用于10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3-5 min。
②无水乙醇脱水2次，每次3-5 min。
③95%的乙醇，3-5 min。
④90%的乙醇，3-5 min。
⑤80%的乙醇，3-5 min。
⑥70%的乙醇，3-5 min。
⑦蒸馏水冲洗2次，每次3-5 min。
2、抗原修复：将切片浸泡在胰蛋白酶抗原修复液中，37 ℃孵育10-25 min。
3、免疫染色洗涤液或自来水洗涤1-2次，每次3-5 min，彻底漂洗干净。
4、进行后续的免疫染色步骤。
(二) 冰冻切片
1、免疫染色洗涤液或自来水洗涤切片5min。
2、抗原修复：将切片浸泡在胰蛋白酶抗原修复液中，37 ℃孵育10-25 min。
3、免疫染色洗涤液或自来水洗涤1-2次，每次3-5 min，彻底漂洗干净。
4、进行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、胰蛋白酶抗原修复液恢复至室温后再使用，但应避免反复冻融。
2、浸泡在胰蛋白酶抗原修复液中，最佳孵育时间需根据不同的样品和目的蛋白自行摸索。
3、为了您的安全和健康，请穿实验服并戴一次性手套操作。
4、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.
Before the advent of heat-induced antigen retrieval, enzyme-mediated epitope retrieval using trypsin, proteinase K, pepsin and other enzymes was the most commonly used method. This trypsin antigen retrieval solution is mainly prepared by dissolving trypsin in a calcium chloride solution with a pH value of approximately 7.8. It can expose antigenic epitopes in samples such as paraffin sections, thereby significantly improving the immunostaining effect. Paraffin sections generally require antigen retrieval treatment, while frozen sections do not necessarily need such treatment. Antigen retrieval can enhance the immunostaining effect of paraffin sections and also improve that of frozen sections to varying degrees; antigen retrieval may be considered when the immunostaining effect of frozen sections is unsatisfactory. Based on the calculation of 10 ml of 1× antigen retrieval solution required per slide, 100 ml of the solution can be used for the antigen retrieval of 10 samples. This reagent is for research use only and not intended for clinical diagnosis or any 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: Immerse the sections in trypsin antigen retrieval solution and incubate at 37 ℃ for 10-25 min.
3. Rinse with immunostaining wash buffer or tap water for 1-2 times, 3-5 min each time, and rinse thoroughly.
4. Perform the subsequent immunostaining steps.
(2) Frozen Sections
1. Rinse the sections with immunostaining wash buffer or tap water for 5 min.
2. Antigen retrieval: Immerse the sections in trypsin antigen retrieval solution and incubate at 37 ℃ for 10-25 min.
3. Rinse with immunostaining wash buffer or tap water for 1-2 times, 3-5 min each time, and rinse thoroughly.
4. Perform the subsequent immunostaining steps.
(3) Other Samples
Operate on other samples with reference to the procedures for paraffin sections or frozen sections.
Precautions
1. Allow the trypsin antigen retrieval solution to return to room temperature before use, and avoid repeated freeze-thaw cycles.
2. When immersing sections in the trypsin antigen retrieval solution, the optimal incubation time should be determined experimentally according to different samples and target proteins.
3. For your safety and health, wear a lab coat and disposable gloves during operation.
4. Use the reagent as soon as possible after opening to prevent adverse effects on subsequent experimental results.
ZJ26F0434136
危险品化学品经营许可证（带存储）