Indirect immunofluorescence microscopy allows two or more antigens to be visualized in the same section, at the same time, and can be used for (1) cell labeling and (2) immunofluorescence screening of stem cells.

Operation method

basic program

Principle

This is done through the use of fluorescent dyes with different excitation and emission wavelengths. The use of two fluorescent dyes is usually limited, most commonly a combination of rhodamine (green excitation, emits red light) and fluorescein (blue excitation, emits green light).

Materials and Instruments

Tissue Samples
Primary Antibody IgG
Slide Humidifier

Move

1. When performing double-labeling experiments, the most important criterion is that the primary antibody should be of a different type so that the secondary antibody can recognize it separately. Preferably, the primary antibodies should be derived from differently immunized animals. However, this requirement may not be met if monoclonal antibodies are used. When monoclonal antibodies are used, different types of antibodies, such as IgG and IgM, can be exactly distinguished by the secondary antibody. However, antibodies of different subclasses (IgG 1 and IgG 2) are usually not exactly distinguished by the secondary antibody.

2. The procedure is the same as the " Immunofluorescence Labeling of Tissue S ections" except that the primary and secondary antibodies are replaced by a mixture of two primary antibodies and two secondary antibodies.3. Before attempting to perform double labeling experiments, single labeling experiments should be performed to map out the optimal dilution of the different primary and secondary antibodies.

Caveat

1. When performing double-labeling experiments, the most important criterion is that the primary antibody should be of a different type so that the secondary antibody can recognize it separately. Preferably, the primary antibodies should be derived from differently immunized animals. However, this requirement may not be met if monoclonal antibodies are used. When monoclonal antibodies are used, different types of antibodies, such as IgG and IgM, can be exactly distinguished by the secondary antibody. However, antibodies of different subclasses (IgG 1 and IgG 2) are usually not exactly distinguished by the secondary antibody.

2. before attempting a double labeling experiment, a single labeling experiment should be performed to feel out the optimal dilution of the different primary and secondary antibodies.

3. Before double staining is performed, separate staining pre-tests must be performed for each of the selected primary antibodies, and the pre-test conditions must be the same as those for double staining. After observing the results of the pre-tests and the correct localization of the antibodies, the double staining experiments will be performed.

Common Problems

Two cases of heavy background coloration when tissue sections are done for immunofluorescence:

I. Background coloring of tissue sections due to the staining process or non-specific fluorescence of the tissue;

Second, background color that occurs due to hardware facilities or viewfinder settings when taking pictures.

For the first problem, the following suggestions are made: 1. use high quality paraformaldehyde fixation to reduce the spontaneous fluorescence of common formaldehyde 2. thin the thickness of the slice 3. rinse the slice under running water after mounting to reduce the fluorescence of impurities 4. rinse well the primary antibody and the secondary antibody (very important) 5. choose high quality sealing medium.

For the second problem, the following suggestions are made: 1. control the exposure time 2. dark room operation.

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