Immunofluorescence (IF/ICC) Tips

Immunofluorescence / IF / ICC assay is a method of immunological detection based on the combination of antibodies and antigens. Immunofluorescence is the method which has fluorescent tag and can be used for immunocytochemistry (ICC) or immunofluorescence (IF) analysis. Immunocytochemistry (ICC) or immunofluorescence (IF) have some difference, immunocytochemistry (ICC) always refers to staining in cells and immunofluorescence (IF) always refers to staining in tissues. Immunofluorescence / IF / ICC has two common protocols according to test principle which include direct and indirect protocol.
There are several microscope designs can be used for analysis of immunofluorescence samples. The simplest is the epifluorescence microscope, and now the confocal microscope is also widely used. With the improvement of technology, various super-resolution microscope designs with much higher resolution can also be used.
There are also some limiting factors for Immunofluorescence / IF/ ICC. One of that is fixing cells, molecules on the outside of the cell membrane or in the supernatant can be bound by the antibodies which allow for living cells to be stained, and molecules in cell membrane must be stained after fixation which may make protein cross-linked and false negative or false positive signals due to non-specific binding.
The specificity of antibody and their antigen is the most important foundation for Immunofluorescence / IF / ICC assay. The antibodies with fluorescent dyes can react with their antigen within a cell and allow visualisation of the distribution of the target within the samples. Immunofluorescence can be used on cultured cell lines, tissue sections, or individual cells. Immunofluorescence may be used to analyze the distribution of proteins, glycans, and small biological and non-biological molecules.