IHC is used by researchers globally to diagnose disease, in drug development and in a wide range of biological research. Identification of cells, components, their origins and pathology can be visualised in glorious colour using IHC in isolation or combined with other techniques. Cellular activity in response to specific chemicals can be observed and quantified to understand efficacy during drug development. Modern IHC techniques allow high-throughput, multiparametric, accurate data analysis at all levels of research.

        IHC技术被研究人员用于诊断疾病，药物开发和广泛的生物学研究。细胞类型，组分，来源和病理的鉴定可以使用IHC独立或与其他技术结合实现其可视化观察。通过在与特定化学物质的反应中，间接地观察和量化细胞活性，以分析药物开发期间的功效。现代的IHC技术允许在各个研究层次进行高通量，多参数，准确的数据分析。

       This sounds straightforward but there are many pitfalls to avoid. Optimizing your IHC protocol will save valuable time and money and produce reliable results. Biorbyt have a wealth of knowledge and experience alongside the perfect solutions to guide your protocol. We are constantly developing our product range to keep you at the forefront of research and our experts are here to support you.

免疫组化的基本步骤：

处理组织样本，福尔马林固定后石蜡包埋，或者速冻包埋。

孵育与目的蛋白特异性结合的抗体。

使用化学底物或荧光染料实现信号在显微镜下的可视化。

       这听起来很简单，但其中有许多陷阱需要避免。优化您的IHC实验方案将可以节省您宝贵的实验时间和成本，并能获得可靠的结果。Biorbyt拥有丰富的IHC技术知识和经验，将帮助您优化出更好的实验方案。我们不断开发我们的产品系列，为您提供科研*前沿的产品和*专业的技术专家支持。

CD38 antibody  orb10319      

 APOBEC3B antibody orb155694

1.Choose the right antibody and Test it 

   选择正确的抗体进行检测

       Review the validated applications listed for the antibodies you are considering, our datasheets will help you. Optimally, your selection should be an antibody that is validated in IHC. Choosing antibodies without IHC validation will introduce an element of risk. To help inform your choices, when selecting antibodies from Biorbyt we make it clear whether the product has been validated in paraffin (IHC-P) or in frozen section (IHP-Fr) and whether it is validated in Western Blot (WB). Biorbyt’s team can recommend which species have been tested and advise you whether a particular antibody might work in a different species based on sequence identity to the immunogen. Once selected, you will need to test the antibody at different concentrations to establish the correct antibody concentration for your experiment. Because diluent choice will affect antibody performance, Biorbyt’s diluents are specifically matched to given antibodies to make things easier.

       查看所使用抗体的已验证实验类型，我们网上的产品信息将为您提供帮助。*佳情况下，您选择应该是经过IHC验证的抗体，使用没有经过IHC验证的抗体将可能引入无法获得良好结果的风险。为了帮助您选择更好的抗体，在biorbyt购买抗体时，我们会确定产品是否在石蜡（IHC-P）或冰冻切片（IHP-Fr）中进行了验证，以及是否在Western Blot（WB）中进行验证。Biorbyt的团队可以推荐哪些物种已经过测试，并根据免疫原序列的同源性预测抗体是否可以在不同物种中反应而提供参考建议。一旦选定抗体，您需要测试不同浓度的抗体以确定实验中最合适的抗体浓度。同时抗体稀释液的选择也会影响抗体的性能，biorbyt可提供与抗体匹配的稀释液，选择我们使您的实验变得更轻松。

2.Preparation of Tissue  样本组织准备

       Either by freezing or fixing in formalin and embedding in paraffin. Your choice is important as both IHC-P and IHP-Fr methods will affect your tissue, there are pros and cons to each method. Freezing tissue will result in morphological changes while embedding in paraffin will cause some shrinkage, creating a falsely high density of antigens.

        组织可以通过固定在福尔马林中，然后石蜡包埋制备石蜡切片或者冷冻包埋制备冰冻切片。 选择石蜡切片（IHC-P）或冰冻切片（IHP-Fr）的方法都有相应的优点和缺点，可以根据您的组织和抗原表达具体情况选择。冷冻组织会导致形态学变化，而石蜡包埋会引起一些收缩，产生高密度假阳性抗原。

3.Control  对照

      The validity and importance of these cannot be emphasised enough. Carrying out controls will lead to a more robust research outcome and make it easy to troubleshoot when unexpected results arise. At every opportunity within your experiment, controls will improve confidence in your data and enable you to see results of interest more clearly. Biorbyt are happy to recommend positive controls to include in your protocol.

      合理对照的有效性和重要性是需要重点强调的。设置对照可以获得更加严谨性的研究结果，并且有助于在试验结果不理想的情况下用于故障排除。在您的每一次实验中，对照设置都有利于提高数据的可信度，并有助于您更清晰明了地分析感兴趣的结果。biorbyt很乐意根据您选择的产品和实验方案给您推荐合理的阳性对照。

4.Tissue Fixation  组织固定

      The solution and methods you choose will affect the quality of staining achieved. The methodology you choose will be dictated by the tissue type, its age as well as the target antigen. Research which methods will suit your sample, factors to consider include: the type and concentration of fixative used, length of time of fixation, pH as well as which additives are used if any. Again, Biorbyt can provide advice.

       实验方案和方法的选择将影响最终切片的染色质量。您选择的方法将取决于组织类型，其年龄以及靶抗原。哪些研究方法适合您的样品，需要考虑的因素包括：所用固定剂的类型和浓度，固定时间，pH值以及使用哪种添加剂。同样的，biorbyt可以根据样本提供具体建议。

5.Antigen Retrieval  抗原修复

       Important to get this right to achieve a good quality staining. Your tissue will need to be treated either with heat or using enzymes to allow your antibody to access your antigen. The use of heat could create artificial changes to your tissue so an appropriate control procedure should be set up alongside. The process of fixation can mask the antigen making binding impossible. In most cases, heating the tissue in an acidic buffer solution is less likely to affect the integrity of the antigen of interest. Biorbyt can guide you, we have the benefit of experience and feedback from our research community about what works best. Ultimately, a good antigen retrieval results in the best staining. Repetition of this stage should also include using an alkaline antigen retrieval buffer.

       抗原修复是获得高质量染色的重中之重。您的组织将需要通过加热或酶处理，以使抗体可以和抗原成功结合。使用热处理进行抗原修复有可能会对组织造成人为改变，因此应该设置相应的对照。组织固定过程会导致抗原决定簇发生交联，而阻止抗原抗体结合。在大多数情况下，使用酸性缓冲溶液进行热处理，可以使抗原决定簇重新暴露，并保持目标抗原的完整性而不影响与抗体结合。经过我们多次研究探索经验，并结合众多客户实验反馈，biorbyt可以助您调整修复方式获得*佳结果。合理的抗原修复方式可以获得*佳的染色结果，当然修复方式的选择也应该包括部分使用碱性抗原修复缓冲液的靶标。

6.Blocking  封闭

      This is the process of blocking unwanted components that your tissue might express such as biotin, alkaline phosphatase and peroxidase which can affect your results. Biotin detection systems could detect endogenous biotin and to avoid this you will need to implement a biotin blocking system. Similarly, if using horseradish peroxidase (HRP) or alkaline phosphatase conjugated secondary antibodies, peroxidases and alkaline phosphatase may be expressed leading to misleading results. To avoid this you should use an IHC tissue primer, Biorbyt can advise you of the best choice to suit your protocol.

       这个过程是封闭组织中表达的非特异组分，例如可能影响您结果的生物素，碱性磷酸酶和过氧化物酶。生物素检测系统可以检测内源性生物素，为了避免这种情况，您需要实施生物素阻断系统。同样，如果使用辣根过氧化物酶（HRP）或碱性磷酸酶标记的二抗，组织表达的过氧化物酶和碱性磷酸酶可能会导致假阳性结果。为了避免这种情况，您应该使用IHC组织内源性酶阻断系统，Biorbyt可以帮您选择*适合您的方案。

7.Non-specific Binding   非特异结合

       Fc receptors, among others, can cause non-specific binding by your antibody. Blocking of these receptors can be done before incubation with the primary antibody. It is important to use a serum from the same species as that used to raise your secondary antibody. Another option is to use an IHC background blocker which will decrease the background signal. This can also be achieved by increasing the blocking time.

        Fc受体等可以引起抗体的非特异性结合，阻断这些受体可以在与一抗孵育之前完成。 我们推荐选择与二抗相同种类的血清进行非特异封闭，或者使用IHC背景阻挡器，可以减少背景信号。延长封闭时间也可以有效地减少非特异性结合。

8.Detection System Choice  检测系统选择

       There are a range of systems available to detect the location and abundance of your protein using a fluorescent dye or chromogenic substrate. For IHC the most frequently used systems are streptavidin-biotin and avidin-biotin. The methodology you have used throughout will influence the effectiveness of your detection system. Perfect methodology could be wasted by the use of a detection system that will not display the results to their full potential. Your choice will depend on your target and its level of expression. Direct detection methods utilise labeled primary antibodies. With indirect detection the primary antibody is unlabeled and a labeled secondary antibody is used that has been raised against the host species of the primary antibody. This indirect method is more commonly used in IHC. Conjugated secondary antibodies can amplify signals so are the preferred option if proteins are of low abundance. If you do have high levels of protein expression, conjugated primaries are a better choice. Your choice of method will depend on your methodology, Biorbyt can provide guidance according to your individual protocol.

       有一系列系统可通过使用荧光染料或显色底物用于检测蛋白质的位置和丰度。对于IHC，常用的系统是链霉亲和素-生物素系统。您选择的检测方法与检测系统是否匹配，会影响最终检测系统是否能有效地发挥其有效性。检测系统的选择取决于目的靶标信息及其表达水平。直接检测方法是利用标记的一级抗体。而间接检测是一抗是未标记的，并且使用针对一抗宿主物种产生的标记二抗，这种间接检测方法在IHC中更常用。因为偶联标记的二抗可以增强信号，所以如果蛋白质的丰度很低，那么间接检测就是*选的选择。如果您确定目标蛋白是高水平表达，直接检测的直标一抗是更好更高效的选择。您选择的检测抗体取决于您的实验方法，Biorbyt可以根据您的实验提供具体指导。

9.Staining  显色

     To achieve an intensity and accuracy of staining, your choice of chromagen needs to be perfect for the tissue section, the target, any other stains being used and the mounting media. As well as staining, counterstaining will increase contrast and perspective. Our wide range of chromogens is designed to provide solutions for every set-up.

       为了达到染色的强度和准确性，染色剂和染色方案要根据组织切片，靶标蛋白，封片剂等进行具选择。除了信号染色，核复染可以增加结果对比度和定位视角的观察。我们提供一系列染色剂旨在为您的每个实验提供*佳解决方案。

IL6 antibody orb6210     

CD133 antibody orb99113

       The Biorbyt team are committed to supporting your research. Use our expertise and experience to create more accurate, robust, and reliable results.

       Biorbyt团队致力于支持您的研究，使用我们专业的IHC知识和经验来帮您创建更准确，更强大，更可靠的结果。