Tissue microarray immunofluorescence (IF) double labeling

Service introduction:

Immunofluorescence technique is a technique established on the basis of immunology, biochemistry, and microscopy. The method of using fluorescent antibodies to trace or detect corresponding antigens is called the fluorescent antibody method; the method of using known fluorescent antigen markers to trace or detect corresponding antibodies is called the fluorescent antigen method. These two methods are collectively referred to as immunofluorescence technique. However, in practice, fluorescent antigen technique is rarely used, so fluorescent antibody technique is often referred to as immunofluorescence technique.

Labeling two antigens on a single section is called immunofluorescence IF double labeling. In conventional immunofluorescence double labeling, the primary antibodies must be derived from different species; if they are the same, the secondary antibodies cannot distinguish them. Different primary antibodies are labeled with different colors and can be used to localize two different antigens. Tissue microarray immunofluorescence can arrange dozens to hundreds of small tissue samples neatly on a single glass slide for staining, keeping experimental conditions consistent across tissue samples on the chip, saving time and reagents, and facilitating unified analysis after scanning. Compared with single-section staining, both time and cost can be reduced. The disadvantage is that the selected tissue area is relatively small, which cannot comprehensively reflect the expression status of individual samples. The tissue microarray immunofluorescence double labeling procedure is basically the same as conventional tissue immunofluorescence double labeling; however, because the samples are relatively precious, the operating procedures are more stringent.

Main applications:

Immunofluorescence staining and imaging analysis are indispensable detection techniques for studying tissue morphology and in situ antigen expression in tissues, and are widely used in various fields of clinical pathology and medical and biological research. Any substance that can act as an antigen or hapten, such as proteins, polypeptides, amino acids, polysaccharides, phospholipids, enzymes, hormones, pathogens, and receptors, can be detected at the cellular and subcellular levels.

Detection principle:

Based on the principle of specific binding between antigen and antibody, immunofluorescence technique first labels known antibodies (or antigens) with fluorophores, and then uses these fluorescent antibodies (or antigens) as probes to detect corresponding antigens (or antibodies) in fixed cells or tissues. When observed under a fluorescence microscope, the fluorophore in the antigen-antibody complex emits fluorescence of a certain wavelength after excitation light irradiation, thereby determining the nature and localization of the target antigen (or antibody). Quantitative techniques (such as flow cytometry) can also be used to determine the content.

Experimental procedure:

Deparaffinization and rehydration - antigen retrieval - serum blocking - primary antibody incubation - secondary antibody incubation - DAPI nuclear staining - mounting - microscopic examination.

Sample requirements and transport conditions:

1. Provide properly fixed and qualified samples. After tissue collection, fix immediately with an appropriate fixative. The volume of fixative is recommended to be more than 10-15 times the tissue volume. Fresh specimens should be fixed in suitable containers for 24-48 h; large specimens should be fixed for 12 h and then cut open and fixed further. Fix samples at room temperature (around 24°C) or refrigerated (4°C); do not freeze or allow ice formation. Fixed samples should be transported at room temperature.

2. Smears and cell climbing coverslips must be fully fixed; paraffin sections must be thoroughly deparaffinized before the experiment.

3. Provide accurate antibody information.

Precautions: