Key issues in immunoelectron microscopy
The combination of immunoelectron microscopy electron microscopy and immunocytochemistry can show the position of antigen or antibody at the level of ultrastructure of cells, which has become an important research tool in the field of biomedicine. Due to the long process of immunoelectron microscopy and the troublesome operation procedures, it is necessary to pay attention to the key links in order to obtain satisfactory immunogold staining. Below we discuss several problems in immunoelectron microscopy. (Related experimental technical services: immunoelectron microscopy technology services)
1. Immobilization of stained specimens <br>The ideal immunoelectron microscopy specimens must be preserved both in the tissue antigen and in the cell ultrastructure. For most antigens, fixing the specimen according to conventional electron microscopy will partially or completely eliminate the antigenicity. Therefore, immunoelectron microscopy usually uses some special fixatives, such as periodate-lysine-paraformaldehyde fixative (PLP). 4% paraformaldehyde fixative, paraformaldehyde plus low concentration of glutaraldehyde fixative (PG) (relevant reagent: glutaraldehyde 25% solution) and picric acid-paraformaldehyde fixative. In our actual work, we found that it is most desirable to add 0.01%-0.05% glutaraldehyde with 1% paraformaldehyde. This fixing solution is not only simple in configuration, but also satisfactory in results. Of course, for some antigenic samples, 4% paraformaldehyde plus 0.05% to 0.5% glutaraldehyde can also be used. Some can even be fixed directly using conventional electron microscopy fixatives.
2. Selection of embedding agent <br>The immunohistochemical staining of the tested tissue before or after embedding should be based on the condition of the labeled antigen. Generally, pre-embedding staining is performed on an antigen that detects only the cell surface, so that there is no destruction agent for dehydration, permeation, and polymerization of the cell antigen. In contrast, post-embedding staining is performed after immunostaining after specimen embedding, which often causes greater damage to the antigen. Therefore, the appropriate embedding agent should be selected according to the nature of the antigen to be tested. In order to reduce the damage to the antigen and increase the positive detection rate. Epon812 is the most commonly used electron microscope embedding agent. It requires the specimen to be thoroughly dehydrated before embedding. Generally, it needs to be polymerized at 60 °C. Therefore, the antigenicity tends to be greatly damaged. We use low-temperature long-term polymerization at work, 45 ° C for 3 days, to reduce the effect of polymerization on antigenic preservation. In order to reduce the destruction of tissue antigenicity and obtain strong positive staining results, many laboratories use low temperature embedding agents, transparent synthetic resins and water-soluble embedding agents (ethylene glycol methyl esters). Low-temperature embedding agents are usually used in post-embedding dyeing of ferritin or colloidal gold immunoelectron microscopy.
3, anti-serum and probe <br> To obtain the ideal immunostaining, the selection of anti-serum with high specificity and affinity is the primary condition for the success of the experiment. The primary antibody used in the general experiment is purchased through the market (click to view: find the antibody), and the purchased primary anti-concentration solution must be diluted with the antibody dilution before use. It is considered that the concentration of the primary antibody used for immunoelectron microscopy after embedding is higher than that of light. The concentration of the mirror stain is 10 to 100 times. One of the reasons why the staining failure sometimes occurs is because the antibody immunohistochemically stained by light microscopy is used as the primary antibody.
Probes are typically used for observation under electron microscopy and are typically displayed using colloidal gold. Colloidal gold probes can be purchased either commercially or by Slot method. The colloidal gold generally prepared has a diameter of 5 nm, 10 nm, and 15 nm.
4, specific adsorption problems <br> immunoelectron microscopy, usually using colloidal gold as a probe, however colloidal gold is a highly sensitive probe, how to overcome the background, improve the specificity of the staining results become colloidal gold immunoelectron microscopy The key link of inspection. The use of high potency, specificity of primary antibody and stable activity, appropriate concentration of immunogold probes and high-quality dilution of appropriate antibodies are important conditions to overcome background contamination and obtain ideal labeling results. As the dilution increases, the effects of contaminating proteins will gradually diminish. Generally, when detecting a new antigen, the dilution of the primary antibody and the gold-labeled antibody must be determined by square matrix titration. In addition, high salt and detergent-containing buffers can be used for elution to reduce background contamination. Usually, sodium chloride and Triton X-100 are added to the buffer to a concentration of 2.5% and 1%, respectively. can. Of course, it is also important that the primary antibody and the gold probe are appropriate. The general time is 24 h (4 ° C) and the gold probe is 2 h at room temperature. In the experiment, we used normal sheep serum (1:50-1:100) or 1% bovine serum albumin as a blocking agent to block non-specific adsorption.
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