(I) Principle Immunoelectron microscopy technology is a product of the combination of immunochemical technology and electron microscopy technology, and is a methodology for studying and observing the binding location of antigens and antibodies at the ultrastructural level. It is mainly divided into two categories: one is the immunocoagulation electron microscopy technology, that is, after using the antigen-antibody agglutination reaction, and then directly observed under the electron microscope by negative staining; This technology is to use a specially labeled antibody to combine with the corresponding antigen, and observe it under an electron microscope. As the standard forms a certain electron density, it indicates the location of the corresponding antigen. The application of immunoelectron microscopy has made the study of the localization of antigens and antibodies into the subcellular level. Stainless Steel Cookware by Fungho add an aesthetic appeal to your stovetop to enhance the overall appearance and functionality of your kitchen. Ellementry Kitchenware,Tasty Kitchenware,Kitchenware Set,Kitchenware Products Fungho Industries (Group) Co., Limited , https://www.funghogroup.com
(2) Some factors affecting immunoelectron microscopy
1. Markers There are three types of markers used for electron microscopy observations: One is the dense electron density markers, such as ferritin and horseradish peroxidase. The other type is radioisotopes, such as 135I, 35S, 32P, 14C, 3H, etc. The third type is uniquely shaped markers, such as hemocyanin, bacteriophage, etc.
The requirements for the markers are: they have a specific shape and do not affect the characteristics and shape of the antigen-antibody complex. The markers currently used in immunoelectron microscopy are mainly ferritin and HRP. Both have their advantages, and the ferritin electron density is dense. Observation has a large contrast, which is better than enzyme labeling, but ferritin has a large molecular weight (460 000) and poor penetration, so it is suitable for the positioning of cell surface antigens. In addition, the labeling process of ferritin is more complicated. HRP has a small molecular weight (40 000) and strong penetrating power, which facilitates the entry of labeled antibodies into cells and is suitable for intracellular antigen localization.
2. Fixing agent fixation is a key step in immunoelectron microscopy. The difference between fixation in immunoelectron microscopy and fixation in ultra-thin sections is to consider not only the preservation of the ultrastructure of cells, but also the problem of antigen inactivation.
(1) Requirements for fixatives: â‘ Do not damage the activity of intracellular antigens; â‘¡ Fast fixation speed and good effect; â‘¢ Small molecular weight, easy to penetrate; â‘£ After fixation, do not cause cross-linking, cause space obstruction and affect labeled antibodies Enter the antigenic position.
(2) The factors affecting the fixation are: ①the type of fixative used; ② the concentration of the fixative is too large, which affects the activity of the antigen, the concentration is too small, the fixation effect is poor; ③ the pH of the fixative; ④ fixation The temperature of the agent is generally cold fixed at 2 ° C to 4 ° C; this can reduce the self-bathing effect of the cells and the extraction of water; ⑤ The fixing time is related to the temperature, the temperature is high, the fixing is fast, and the ionic strength of the buffer system, Large ionic strength, high osmotic pressure, strong penetrating power, and fast fixation. The fixation time required for different fixatives or different concentrations of the same fixative is also inconsistent; ⑥ It is related to the type of cells to be fixed.
The current commonly used fixed systems are: 4% polyoxymethylene, 1.5% to 2% glutaraldehyde, 1% paraformaldehyde + 1% glutaraldehyde, 4% paraformaldehyde + 0.5% picric acid + 0.25% glutaraldehyde , 96% ethanol + 1% acetic acid. Regardless of the system used, a series of pre-tests must be done with antigens of known potency before use. Such as the concentration, temperature, pH and fixed time of the fixing agent. Then make the pretreatment titer and use it as a reference for inactivation to select the most suitable conditions.
3. Non-specific adsorption Non-specific adsorption is related to the dilution of enzyme-labeled antibody and antiserum, staining time, temperature and medium, among which the most important is the dilution of antiserum and labeled antibody. It is generally believed that high-potency antiserum or labeled antibody is diluted to a low protein concentration for optimal staining results. Because low protein concentration is beneficial to reduce non-specific adsorption. The practical protein concentration is roughly 0.50mg / ml ~ 2mg / ml. The work titer is generally 1: 20 ～ 1: 400. In actual work, diluting the labeled antibody or antiserum to more than 1: 100 times can obtain the ideal positive result, and the non-specific adsorption must be reduced very low.
The choice of working concentration is to dilute the labeled antibody or antiserum 1: 2, 1: 4, 1: 8 ... 1: 256, do the staining observation of the known positive specimen, and take the positive deposits as obvious, not special The lowest dilution of heterosexual adsorption is used as the working concentration.
4. Marking staining method Marking staining method is divided into direct staining method and indirect staining method. The former is characterized by high specificity and low sensitivity, and labeled antibodies can only be used to detect one antigen. The latter is more sensitive, and one labeled antibody can be used for the detection of multiple antigens. The disadvantage is poor specificity