The monoclonal antibody has the best binding effect when the antigen can maintain its natural conformation. Direct detection is not disturbed by non-specific binding of secondary antibodies, and the operation is simpler when performing multi-color detection. Most cells only need to remove the medium and then add a fixative. However, blocking and control were added due to the need to bind secondary antibodies.
Therefore, cryo tubes with biotinylated secondary antibody and avidin or streptavidin can significantly amplify the signal. The fixation principle is to compete with water for hydrogen bonds in proteins, thereby replacing water molecules in tissues and stabilizing the secondary structure while affecting the tertiary structure of the protein. Multicolor experiments must be designed to limit the cross-reactivity between the detection reagents and the spectral properties of the fluorescent dyes used.
There are many steps for paraffin sectioning, and the antigen activity in the preparation is reduced, but the tissue structure and cell morphology are clear. After freezing and slicing, fix the slices with ethanol to prevent the epitope from being cross-linked by formaldehyde, without the need for antigen repair.
After two minutes, remove the pre-fixation medium and add fresh 2% fixative to continue the fixation. DAB is more commonly used than AEC because the latter is easily soluble in alcohol, and it is more likely to fade when exposed to excessive light. It is not recommended to perform antigen repair after alcohol fixation to avoid damaging the integrity of the sample. Alcohol fixatives commonly used are alcohol and ethanol. Heat-induced repair vs enzyme-induced repair Heat-induced epitope repair (HIER) needs to consider factors such as antigen, antibody, tissue type, fixation method, and duration in the antigen repair process. It is a conventional preparation method for immunohistochemistry.
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