Immunohistochemical Antibody Selection Points
Selection of Primary Antibody
1. Choose monoclonal or polyclonal antibody? A single specific antibody produced by one type of B cell is called a monoclonal antibody. A monoclonal antibody binds to a single specific antigenic determinant in a well-targeted manner, like a missile hitting its target precisely. On the other hand, even for the same antigenic determinant, antibodies can be produced by several types of B cells in the body, forming antibody hybrids called polyclonal antibodies. In antigen-antibody reaction, generally monoclonal antibodies have strong specificity, but relatively small affinity and relatively low sensitivity in detecting antigens; whereas polyclonal antibodies have slightly weaker specificity, strong affinity and high sensitivity, but are prone to non-specific staining (non-specific staining can be avoided as much as possible during the experiment by closure, etc.).
2. The source of antibody. Generally, rabbit-derived antibodies are polyclonal, while mouse-derived antibodies are monoclonal, but there are exceptions. The source of the antibody is very important, and the labeled secondary antibody must match the primary antibody.
3. What kind of antigen is to be detected, i.e., Species Reactivity, which is very important and generally indicated on the instruction manual, such as Mouse, Rat, Human, etc. Try to choose the antibody whose reactivity has been indicated on the instruction manual when purchasing.
4. Can we do immunohistochemistry? Generally, the specification of the primary antibody will indicate whether it can do WB, IHC, ICC, IF, etc. It is recommended that the best choice of the antibody indicated, because it is generally verified by experiments.
5. Testing specimen type. Whether the user is detecting paraffin sections or frozen sections, generally antibodies that can do paraffin sections can be used to detect frozen sections; but antibodies that can do frozen sections may not be able to detect antigens in paraffin sections.
6. Price and quality. Imported antibodies are long and expensive, and many of them are OEM from domestic manufacturers. Antibodies for scientific research are concentrated antibodies, which need to be tested several times to establish suitable experimental conditions before conducting large-volume experiments, and the experimental operator's proficiency and control of experiments have a great impact on the experimental results, so the experimental details must be well controlled. In addition, the antibody should avoid repeated freezing and thawing.
Selection of secondary antibodies
1. Genus source. It is mainly based on the source of the primary antibody to decide to buy the source of the secondary antibody, such as the primary antibody is a mouse source, then the secondary antibody can be bought anti-mouse (the source of the secondary antibody goat or Rabbit can be); such as the primary antibody is a rabbit source, then the secondary antibody can be purchased to anti-rabbit.
2. Selection of markers. Common markers include HRP, Biotin, fluorescein and so on. Immunohistochemistry at this stage mostly uses two-step method, the secondary antibody used in two-step method is usually labeled with poly-HRP, and the effect of ordinary HRP labeling applied to immunohistochemistry will be relatively poor; the three-step method, which was applied more in the previous years, has Biotin labeling on the secondary antibody, and then HRP-labeled streptomycin is added for further reaction. Immunofluorescence staining requires the purchase of fluorescein-labeled secondary antibody, such as rhodamine, FITC, Cy3 and other commonly used fluorescein. How to choose the secondary antibody mainly depends on whether the experimental application is immunohistochemistry or immunofluorescence.
3. choose IgM or IgG. the secondary antibody needs to match the class or subtype of the primary antibody, which is usually for monoclonal antibodies. Polyclonal antibodies are mainly IgG immunoglobulins, so the corresponding secondary antibody is an anti-IgG antibody.
The class and subtype of the monoclonal antibody is usually listed in the product listing. If your primary antibody is a mouse IgM, then the corresponding secondary antibody should be either an anti-mouse IgM, or an anti-mouse IgG antibody. If the monoclonal primary antibody is a mouse IgG subtype (IgG1, IgG2a, IgG2b, IgG3), then almost all anti-mouse IgGs will bind to it, or you can choose a secondary antibody that is specific to that subtype, e.g., if your primary antibody is a mouse IgG1, then you can choose an anti-IgG1 secondary antibody, which is particularly suitable for double-labeling experiments. This antibody is particularly suitable for double labeling experiments. If you don't know which class or subtype the primary antibody is, then anti-mouse IgG is a good choice, as this antibody recognizes most types of IgG immunoglobulins.