免疫组织化学和免疫细胞化学：通过光显微镜进行组织成像
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Overview
资料来源：迈克尔·李1和托尼娅·韦伯1
1马里兰大学医学院微生物学和免疫学系，马里兰州巴尔的摩的马琳和斯图尔特·格林鲍姆综合癌症中心 21201
免疫组织化学 （IHC） 和免疫细胞化学 （ICC） 是使用抗体可视化特定抗原的表达和定位的技术。IHC的首次使用是在1941年，当时阿尔伯特·库因斯利用这项技术来可视化感染肺炎球菌（1）的小鼠在组织部分中存在肺炎球菌抗原。名称，免疫组织化学，是从根”免疫-“，指抗体，和”组织-“，参照IHC中使用的组织部分。免疫细胞化学中根的”细胞-“突出了ICC和IHC之间的关键区别。IHC使用整个组织的各个部分，而ICC使用从组织分离或培养的细胞。所用样品的差异意味着样品制备在技术上因IHC和ICC而异，但除此之外，ICC和IHC的协议是相同的，人们会发现这些术语是可互换使用的。
在IHC和ICC中，分别带有化学或荧光标记的抗体，如过氧化物酶或罗丹，通过标记的抗体与抗原的特异性结合，用于可视化任何感兴趣的抗原的分布。在IHC的情况下，薄薄的组织切片被固定在幻灯片上，在被染色之前保持组织的结构，从而允许在整个组织环境中显示抗原（图1）。在ICC的情况下，细胞在被染色之前均匀地分布在幻灯片上，允许在单个细胞内显示抗原分布，但不在任何特定组织的结构内。由于两个协议之间的相似性，该协议将侧重于IHC，以解决IHC中涉及样品制备的额外复杂性。
图1：IHC协议大纲。从小鼠解剖的石蜡嵌入组织的 IHC 协议的视觉轮廓。该协议使用生物回位抗体和链球菌-HRP来可视化抗体结合的位置。其他选项，如荧光标记抗体，也是可能的。请点击此处查看此图的较大版本。
执行 IHC 时的第一个重大决策是如何准备组织部分，以便在整个染色过程中保持组织的结构。两个主要选择是石蜡嵌入组织或冷冻组织的新鲜部分的正式固定部分。对于使用哪种方法，没有简单的答案，因为它取决于将进行的下游分析。石蜡嵌入组织的形式固定通常被认为可以更好地保存组织形态，以实现最佳成像，而冷冻新鲜组织可以保留蛋白质功能，用于 IHC 以外的后续检测。此外，新鲜冷冻组织部分已被证明更适合基因表达分析（2）。第三个考虑因素是，您感兴趣的抗原的抗体是否适合固定或冷冻组织部分，因为某些抗体仅针对特定类型的部分进行了优化，可能不适用于其他部分。最后，还需要确定它们需要多久来储存组织部分，因为新鲜冷冻样品必须保持在-80°C，并且不能持续超过一年，而固定切片可以在室温下储存更长时间。这些是确定是使用石蜡嵌入组织的形式固定部分还是冷冻组织的新鲜部分的一些主要考虑因素。最终，如果一个人有足够的组织，最好只是有一些两者。
在这个实验中，我们开始确定环素D1表达是否从淋巴瘤发育的自发小鼠模型增大了扩大的脾脏。首先从野生型小鼠、没有淋巴瘤的转基因小鼠或自发发展淋巴瘤的转基因小鼠中分离出脾组织样本。脾脏组织样本固定在甲醛中，嵌入石蜡中，分片，使用小鼠抗环素D1原抗体染色，然后使用马抗小鼠二次抗体，并使用3，3-二氨基苯甲酸（DAB）开发。然后，在哈里斯赫马图林溶液中对节进行反染色，然后以20倍放大倍率对各部分进行成像。
试剂
石蜡嵌入部分
- 4% 甲醛 （PFA）
- 乙醇（无水变性，组织学等级100%，95%，80%，75%，和50%）。可使用双蒸馏水（ddH2O）从100%库存中稀释
- 二甲苯
- IHC 兼容玻璃滑动，以确保组织部分在整个过程中保持连接。IHC 兼容玻璃玻片具有专用涂层，可从多个零售商处随时获得。如果执行 ICC，请使用室幻灯片。室室幻灯片允许将细胞播种在腔室中并放置在培养箱中，直到细胞附着在幻灯片上并达到适当的汇合点，此时，室可以去除，染色方式可以像 IHC 一样进行。
- 石蜡
- 0.3% 过氧化氢 （H2O2）/甲醇：要制备，添加 1 mL 30% H2O2至 99 mL 甲醇。储存在-20°C
- 抗原检索缓冲液：IHC酸酸缓冲液pH6.0
新鲜冷冻部分
- 最佳切削温度 （OCT） 嵌入化合物
- 最佳固定性：4% PFA 或丙酮已冷却至 -20°C
染色
- 阻塞缓冲区：应由用户确定。一个例子是马血清在1XPBS中稀释
- 稀释原抗体：参见制造商规格
- 稀释的生物防化二级抗体：参见制造商规格
- 稀释的红萝卜过氧化物酶（HRP）：仅适用于过氧化物酶可视化。请参阅制造商规格。
- DAB 或其他兼容基板
- 反污（可选）
- 乙醇（无水变性，组织学等级100%和95%）
- 二甲苯
- 奥诺/利莫内山
Procedure
Results
IHC and ICC have a vast range of applications. For example, one use of IHC is to examine the expression of oncogenes in spontaneous mouse models of tumor development. In Figure 2, we set out to determine if cyclin D1 expression was increased in enlarged spleens in a spontaneous mouse model of lymphoma development. Splenic tissue samples were fixed in paraformaldehyde, embedded in paraffin, sectioned, stained using an anti-cyclin D1 antibody (diluted 1:200 in blocking buffer), and then the sections were imaged at 20X magnification. Cyclin D1 expressing cells are indicated by the reddish-brown color against the blue tissue background. These results suggest that cyclin D1 expression was increased in enlarged spleens, indicating a correlation between cancer development and cyclin D1 expression in this model.
Figure 2: Splenic Cyclin D1 Expression in a Spontaneous Double Transgenic (DTG) Mouse Model of Lymphoma. An image of splenic tissue stained with an anti-Cyclin D1 primary antibody, counterstained with methyl green, and visualized using a biotinylated secondary antibody and ABC reagent activated with DAB substrate. The reddish-brown color represents locations where the antibody has bound indicating the presence of Cyclin D1 expressing tumor cells within the structure of splenic tissue that has been counterstained blue. Please click here to view a larger version of this figure.
Applications and Summary
Immunohistochemistry (IHC) and immunocytochemistry (ICC) are techniques used to visualize the expression and localization of specific antigens using antibodies. Tissues are first cut into thin sections that maintain the tissue morphology and placed on a slide. The antibodies are then added and will bind the antigen of interest and are equipped with a specific tag that allows them to be visualized under a microscope. Thus, through this basic concept, the distribution of antigens in the context of tissue structure can be visualized and studied. However, while the overarching concept is basic, there are multiple different approaches and variations that have been developed that increase both the complexity and usefulness of these techniques. This paper has covered the basic concept of IHC and ICC, the main decisions that need to be considered when using these techniques, and a detailed step-by-step protocol. The images produced by IHC and ICC are generally the final product and can be published as is to highlight obvious differences in amounts or distribution of staining between different conditions.
References
- Coons, A. H. Creech, H. J., Jones, N. and Berliner, E. The Demonstration of Pneumococcal Antigen in Tissues by the Use of Fluorescent Antibody, The Journal of Immunology, 45 (3), 159-170 (1942).
- Ripoli, F. L., Mohr, A., Hammer, S. C., Willenbrock, S., Hewicker-Trautwein, M., Hennecke, S., Escobar, H. M. and Nolte, I. A comparison of fresh frozen vs. Formalin-fixed, paraffin-embedded specimens of canine mammary tumors via branched-DNA assay. International Journal of Molecular Sciences, 17 (5) (2016).
Transcript
Immunocytochemistry and immunohistochemistry are staining methods for a protein of interest in cultured cells and tissues, respectively. The basic principle of both related techniques involves using specific antibodies tagged with a detection system to identify and visualize the protein and determine its location within the cells and tissues, as well as the relative levels. The process in either experiment begins with sample preparation.
For immunocyctochemistry, which specifically visualizes protein or antigen locations in cells, this involves three steps. The first step is plating, which entails culturing the cells in growth media on a cover slip or slide, typically, in the wells of a culture plate. This is followed by fixation, where a precipitating or crosslinking agent like paraformaldehyde is added to the cells to preserve the structural integrity of the proteins and prevent enzyme activity from degrading them. The last step is permeabilization, which involves adding a detergent to make the cell membranes permeable for the staining.
In the counterpart method, immunohistochemistry, proteins or antigens are visualized in tissues and sample preparation has five steps. First, the whole tissue is subjected to fixation, usually with paraformaldehyde. This is followed by embedding of the tissue in a block of paraffin, and then sectioning of this block using a machine called a microtome to cut the tissue into thin slices which can be placed onto slides. Next, the slides are subjected to deparaffinization, or removal of the paraffin from around the tissue slice. Then, an optional antigen retrieval step can be performed. This can either be done using heat or enzymes to unmask epitopes that were cross-linked during fixation making them available for antibody binding. After the appropriate sample preparation, a target-specific primary antibody is added to the cell or tissue sample. This primary antibody should bind to the protein of interest. Next, a secondary antibody is added, which detects and binds to the primary antibody. This secondary antibody is conjugated to, or can bind to, an enzyme called HRP. When its specific substrate, DAB, is added, HRP converts this to an insoluble, brown precipitate. This brown stain marks the location of the target protein. The slides are also stained with hematoxylin, which labels the nuclei in blue and provides a spatial reference point for determining subcellular localization. After that, mounting media is added to the slide, followed by a cover slip in order to seal and preserve the stained sample. Finally, the slides can be imaged on a light microscope.
In this video, you will observe the sample preparation technique for plated cells and tissue sections, followed by immunostaining of the tissue sections.
First, the cells of interest need to be seated onto coverslips. To do this, working in a tissue culture hood, place individual coverslips into the wells of a 24-well plate. Then, close the sash and turn on the UV light to sterilize the coverslips for at least 15 minutes. Next, turn off the UV light. To lift the cells of interest from a confluent 10-centimeter dish, aspirate the media, wash briefly with PBS, and add trypsin to the cells for 2 minutes. Then, tap the side of the plate to ensure the cells have detached and neutralize the trypsin with media. Next, add 0. 5 mL of the cell suspension into each well, making sure to cover the coverslips. Place the plate into a humidified CO2 incubator and allow the cells to grow at 37 degrees celsius until they are 50-70% confluent.
Once the cells reach the optimal confluency, aspirate the culture medium from each well, and then fix the cells by incubating them in . 5 mL of 4% paraformaldehyde diluted in 1X PBS for 20 minutes at room temperature. After removing the fixative, rinse the cells be adding 1 mL of 1X PBS over each coverslip. Immediately aspirate the PBS, then repeat the rinse 2 more times for a total of 3 washes.
Now, permeablize the cells by adding 0.5 mL of 0.1% Triton X-100 in 1X PBS to each well. Leave the plate at room temperature for 15 minutes. Aspirate off the permeabilization buffer and then rinse the cells by adding 1 mL of 1X PBS into each well. Immediately aspirate off the PBS and repeat the rinse 2 more times for a total of 3 washes. Now that the cells on the coverslips are fixed and permeabilized, proceed to the staining procedure demonstrated for the following immunohistochemistry example with the exception that the incubations should be performed within the wells of the 24-well plate rather than directly on a tissue section slide.
To begin, obtain prepared, formalin-fixed, paraffin-embedded tissue sections. Deparaffinize the slides by placing them into a slide rack and then completely immersing them into 250 mL of 100% xylene. Allow the slides to incubate for 5 minutes in the xylene. Then, remove the slides from the container, wipe them off with a paper towel, and place them into a new xylene bath in a fresh container for a further 5 minutes.
Next, rehydrate the sections in a series of graded ethanol solutions starting with 100% ethanol for 3 minutes. Wipe off the slide rack with a paper towel and transfer the slides to a new container of 100% ethanol for another 3 minutes. Continue this cycle of washing, drying with a paper towel, and transferring the slides to a new bath following the indicated concentrations of ethanol for the specified time. After the final ethanol wash, wipe off the rack with a paper towel and incubate the slides in 100 mL of .3% hydrogen peroxide for 30 minutes at room temperature in order to block any endogenous peroxidase activity. Wash the slides in 250 mL of 1X PBS for 5 minutes. Repeat this wash in a container of fresh 1X PBS for an additional 5 minutes.
Next, perform antigen retrieval by immersing the slides in 250 mL of IHC citrate buffer at pH 6.0 and boiling them for 20 minutes. Then, proceed to the staining protocol.
To begin the staining process for IHC, circle the sections with a hydrophobic pen to identify the minimal area that the buffer needs to cover. Then, use a pipette to place 100 microliters of blocking buffer, which in this experiment is horse serum diluted in 1X PBS, over the section. Incubate the slides for 1 hour at room temperature. Following this, remove the blocking buffer using a pipette.
Next, dilute the primary antibody and blocking buffer at a 1:100 dilution by adding 990 microliters of horse serum diluted in 1X PBS into a 1. 5 mL Eppendorf tube, followed by 10 microliters of the primary antibody. Add 100 microliters of the diluted primary antibody to each section, and incubate the slides for 30 minutes at room temperature. When the timer sounds, drain the primary antibody off each slide, and then wash them in 250 mL of 1X PBS for 5 minutes. Repeat this wash once more using fresh 1X PBS.
While the slides are washing in 1X PBS, dilute the secondary antibody to a 1:200 dilution by adding 995 microliters of blocking buffer to a 1.5 mL tube followed by 5 microliters of the secondary antibody, which in this case is biotinylated horse anti-mouse IGG. Add 100 microliters of the diluted secondary antibody to each section, and then incubate the slides for 30 minutes at room temperature. After 30 minutes, remove the secondary antibody by draining it off the sections, then wash the slides in 250 mL of 1X PBS for 5 minutes. Repeat this wash using fresh 1X PBS.
Now, add 100 microliters of avidin-biotin complex reagent, and incubate the sections in the dark for 30 minutes at room temperature. Next, wash the slides by immersing them in 250 mL of 1X PBS for 5 minutes. Similar to previous wash steps, repeat this wash one more time using fresh 1X PBS. Next, develop the slides by incubating the sections in 100 microliters of DAB for up to 5 minutes. Stop the development by immersing the sections in 250 mL of distilled water for 5 minutes.
Now, slides can be counterstained, if desired. To do this, briefly dip the slides in 250 mL of Harris Hematoxylin Solution. Rinse off the counterstain by washing the slides in 250 mL of distilled water for 5 minutes. Repeat this wash 1 more time using fresh distilled water. Next, dehydrate the sections. To do this, first incubate the slides in 95% ethanol for 5 minutes. Blot the slides on a paper towel, and transfer them to a new container of fresh 95% ethanol for another 5 minutes. Continue the cycle of washing, blotting with a paper towel, and transferring the slides to a new bath, following the indicated solutions for 5 minutes each.
After the final incubation, blot the slides with a paper towel, then add a drop of mounting media, such as Organo-Limonene Mount, to the slides. Now, place a coverslip over the sections, taking care not to trap air bubbles. The slides are now ready to be observed under a microscope for analysis.
To observe the stained sections, use a standard light microscope to visualize the stain, and a digital camera to capture the image. In this particular example of IHC, spleen tissues from wild type and spontaneous, double-transgenic, or DTG mice, are compared for studying Dyclin D1 expression in lymphoma. The tissues were paraffin-embedded, sectioned, and stained with anticyclin D1 antibody, and imaged at 20X magnification. Cyclin D1 expressing cells are indicated by the reddish-brown color against the blue tissue background. Comparing the staining intensities among the images from the various mice, the non-enlarged spleens have relatively low amounts of Cyclin D1 expression irrespective of the mouse genotype. In contrast, the enlarged spleen from the DTG mouse, shows increased reddish-brown staining indicating a correlation between cancer development and Cyclin D1 expression in this mouse model.