免疫组化方法的具体步骤

免疫组化方法的具体步骤
英文回答：
Immunohistochemistry (IHC) is a widely used technique in biomedical research and clinical diagnostics to detect and visualize specific proteins or antigens in tissue samples. The technique involves several steps to achieve accurate and reliable results.
1. Tissue preparation: The first step in IHC is to prepare the tissue sample. This involves fixing the tissue in a suitable fixative, such as formalin, to preserve its structure and prevent degradation of proteins. The fixed tissue is then embedded in paraffin or frozen for sectioning.
2. Sectioning: The tissue sample is cut into thin sections using a microtome. Paraffin-embedded samples are sliced into thin sections (usually around 4-6 micrometers thick), while frozen samples can be sectioned even thinner
(around 10-20 micrometers thick). These sections are mounted onto glass slides for further processing.
3. Deparaffinization and rehydration: If the tissue sample was embedded in paraffin, the sections need to be deparaffinized to remove the wax. This is typically done by soaking the slides in xylene or a xylene substitute, followed by a series of alcohol washes to rehydrate the tissue.
4. Antigen retrieval: In some cases, the antigens of interest may become masked or cross-linked during the fixation process, making them inaccessible to antibodies. Antigen retrieval methods, such as heat-induced epitope retrieval or enzymatic digestion, are used to unmask the antigens and improve their detection. This step is particularly important for formalin-fixed paraffin-embedded (FFPE) samples.
5. Blocking: To prevent non-specific binding of antibodies, the tissue sections are incubated with blocking solutions. These solutions usually contain proteins, such
as bovine serum albumin (BSA) or milk, which occupy any available binding sites on the tissue and reduce background staining.
6. Primary antibody incubation: The primary antibody, specific to the target antigen, is applied to the tissue sections and allowed to bind to the antigen of interest. The primary antibody can be monoclonal or polyclonal, depending on the experimental requirements. The incubation time and temperature may vary depending on the antibody and tissue type.
7. Washing: After the primary antibody incubation, the tissue sections are washed to remove any unbound antibodies or other reagents. This step helps to reduce background staining and improve the specificity of the staining.
8. Secondary antibody incubation: A secondary antibody is applied to the tissue sections, which recognizes and binds to the primary antibody. The secondary antibody is conjugated to an enzyme, fluorophore, or other detection molecule, allowing for visualization of the antigen-
antibody complex.
9. Washing: Similar to the previous washing step, the tissue sections are washed to remove any unbound secondary antibodies.
10. Visualization: Depending on the detection molecule used, different visualization methods can be employed. For enzyme-conjugated secondary antibodies, a chromogenic substrate is added, resulting in the formation of a colored precipitate at the site of antigen-antibody binding. Fluorescently-labeled secondary antibodies can be
visualized using fluorescence microscopy.
11. Counterstaining and mounting: To enhance the visibility of the stained tissue, counterstaining with dyes like hematoxylin or eosin can be performed. After counterstaining, the tissue sections are dehydrated and mounted with a coverslip using a mounting medium.
中文回答：
免疫组化（Immunohistochemistry，简称IHC）是一种在生物
医学研究和临床诊断中广泛使用的技术，用于检测和可视化组织样
本中的特定蛋白质或抗原。

该技术涉及多个步骤，以获得准确可靠
的结果。

1. 组织制备，IHC的第一步是准备组织样本。

这涉及使用适当
的固定剂（如福尔马林）来固定组织，以保持其结构并防止蛋白质
降解。

固定的组织随后被包埋在石蜡中或冷冻以进行切片。

2. 切片，组织样本使用显微切片机切成薄片。

石蜡包埋样本切
成薄片（通常约4-6微米厚），而冷冻样本可以切得更薄（约10-
20微米厚）。

这些切片被安装在玻璃载玻片上以进行进一步处理。

3. 脱脂和复水，如果组织样本是包埋在石蜡中，需要去除蜡质。

通常通过将载玻片浸泡在二甲苯或替代品中，然后进行一系列的醇
洗涤来复水组织。

4. 抗原修复，在某些情况下，抗原可能在固定过程中被掩蔽或
交联，使其对抗体不可及。

抗原修复方法（如热诱导表位修复或酶
消化）用于解除抗原的掩蔽并改善其检测。

这一步对于福尔马林固
定的石蜡包埋（FFPE）样本尤为重要。

5. 阻断，为了防止抗体的非特异性结合，组织切片与阻断溶液一起孵育。

这些溶液通常含有蛋白质（如牛血清白蛋白或牛奶），它们占据组织上的任何可用结合位点，并减少背景染色。

6. 一抗孵育，将特异于目标抗原的一抗应用于组织切片，并允许其与感兴趣的抗原结合。

一抗可以是单克隆或多克隆的，具体取决于实验要求。

孵育时间和温度可能因抗体和组织类型而异。

7. 洗涤，一抗孵育后，对组织切片进行洗涤以去除任何未结合的抗体或其他试剂。

这一步有助于减少背景染色并提高染色的特异性。

8. 二抗孵育，将二抗应用于组织切片，二抗能够识别并结合一抗。

二抗与酶、荧光染料或其他检测分子结合，从而可视化抗原-抗体复合物。

9. 洗涤，类似于前面的洗涤步骤，对组织切片进行洗涤以去除任何未结合的二抗。

10. 可视化，根据所使用的检测分子，可以采用不同的可视化方法。

对于与酶结合的二抗，会添加一种染色底物，导致抗原-抗体结合位点形成有色沉淀物。

荧光标记的二抗可以使用荧光显微镜进
行可视化。

11. 对比染色和封片，为了增强染色组织的可见性，可以使用血红素或嗜酸染料等染料进行对比染色。

对比染色后，组织切片被脱水并使用封片剂装在玻片上。

以上是免疫组化方法的具体步骤。

通过固定、切片、脱脂、抗原修复、阻断、一抗孵育、洗涤、二抗孵育、洗涤、可视化、对比染色和封片等步骤，可以检测和可视化组织样本中的特定蛋白质或抗原。

这一技术在研究和临床实践中起着重要的作用，有助于我们了解疾病的发生机制和诊断疾病。