(1) Principle
Immunoelectron microscopy is the product of the combination of immunochemical technology and electron microscopy, and is a methodology for studying and observing the binding localization of antigens and antibodies at the ultrastructural level. It is mainly divided into two categories: one is immune agglutination electron microscope technology, that is, after using antigen-antibody agglutination reaction, it is directly observed under electron microscope by negative staining; the other is immune electron microscope localization technology.
This technology uses a specially labeled antibody to combine with the corresponding antigen, and observes it under an electron microscope. Because the standard forms a certain electron density, it indicates the location of the corresponding antigen. The application of immunoelectron microscopy enables the study of antigen and antibody localization to enter the subcellular level.
(2) Some factors affecting immunoelectron microscopy
1. Markers
There are three types of markers for electron microscopy: one is electron-dense markers, such as ferritin, horseradish peroxidase, etc. The other type is radioisotopes, such as 135I, 35S, 32P, 14C, 3H, etc., and the third type is a marker with unique shape, such as hemocyanin, bacteriophage, etc.
The requirements for the label are: it has a specific shape and does not affect the characteristics and shape of the antigen-antibody complex. At present, the main markers for immunoelectron microscopy are ferritin and HRP. Both have their own advantages, and ferritin has dense electron density. The contrast of observation is large, which is better than that of enzyme labeling, but the molecular weight of ferritin is large (460 000) and its penetration ability is poor, so it is suitable for the localization 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 is conducive to the entry of labeled antibodies into cells and is suitable for intracellular antigen localization.
2. Fixative
Fixation is a key step in immunoelectron microscopy. The difference between fixation in immunoelectron microscopy and general ultrathin sectioning is that it considers both the preservation of the ultrastructure of cells and the inactivation of antigens.
(1) Requirements for fixatives:
①Do not damage the activity of intracellular antigens;
②The fixing speed is fast and the effect is good;
③ Small molecular weight, easy to penetrate;
④ After fixation, it will not cause cross-linking, cause space obstruction, and affect the entry of labeled antibody into the antigenic site.
(2) The factors that affect the fixation are:
①The type of fixative used;
②The concentration of the fixative, if the concentration is too large, it will affect the activity of the antigen, and if the concentration is too small, the fixation effect is poor;
③ pH of the fixative;
④The temperature of the fixative is generally 2°C-4°C cold fixation; this can reduce the self-bath of the cells and the extraction of water;
⑤ The fixation time is related to the temperature. The high temperature and the fast fixation are also related to the ionic strength of the buffer system. The ionic strength is large, the osmotic pressure is large, the penetration force is strong, and the fixation is fast. Different fixatives, or different concentrations of the same fixative, require different fixation times;
⑥It is related to the fixed cell type.
The most commonly used fixing systems are:
4% polyoxymethylene, 1.5%-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 preliminary experiments with antigens of known titers must be performed before use. Such as the type of fixative concentration, temperature, pH and fixation time. The pretreated titer is then made as a reference for inactivation to reselect optimal conditions.
3. Nonspecific adsorption
Nonspecific adsorption is related to enzyme-labeled antibody, dilution of 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-titer antiserum or labeled antibody diluted to a low protein concentration can obtain the best results for labeled staining. Because low protein concentration is beneficial to reduce non-specific adsorption. The practical protein concentration is approximately 0.50mg/ml-2mg/ml. The working titer is generally 1:20-1:400. In practical work, ideal positive results can be obtained by diluting the labeled antibody or antiserum to more than 1:100 times, but the non-specific adsorption is bound to be very low.
The choice of working concentration is to dilute the labeled antibody or antiserum by 1:2, 1:4, 1:8… The dilution with the lowest anisotropic adsorption was taken as the working concentration.
4. Marker Staining
There are two types of marker staining methods: direct staining and indirect staining. 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 that the specificity is poor.
