Immunofluorescence technology, also known as fluorescent antibody technology, is one of the earliest immunofluorescence techniques in marker immunoassay. It is a technology built on the basis of immunology, biochemistry and microscopy.
Experimental method Immunofluorescence localization method Experimental principle Immunofluorescence cytochemistry is the detection of target antigens (or antibodies) in the tissue and cell samples to be tested using fluorescein-labeled known antibodies (or antigens) as probes. The antibody complex carries fluorescein. Under the fluorescence microscope, the fluorescein emits bright fluorescence due to the ultraviolet light of the high-pressure mercury lamp source, so that the position and properties of the antigen (or antibody) can be distinguished, and Fluorescence quantification technology is used to calculate the content of antigen to achieve the purpose of localization, qualitative and quantitative determination of antigenic substances.
Experimental material nuclear protein
Reagent, kit methanol PB primary antibody secondary antibody
Instruments, consumables, microscope slides, coverslips, dropper
Experimental Procedure 1. Cells were cultured on a 22 x 22 mm 1.5 coverslip. Under ideal conditions, cells should grow to 50% to 70% confluence.
2. Fix the cells with 100% methanol for 3 minutes at -20 °C.
3. Wash three times with PBS + 1% NGS for 10 minutes each time.
4. Incubate for 1 hour at room temperature with a suitable concentration of primary antibody in a wet box.
5. Wash three times with PBS + 1% NGS for 10 minutes each time.
6. Incubate in a wet box with a fluorescently labeled secondary antibody diluted to 4 ug/ml for 1 hour at room temperature.
7. Wash four times with PBS for 10 minutes each time.
8. Seal the coverslip on the slide with a sealing plate and seal the coverslip with clean nail polish to prevent the coverslip from sliding.
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Note 1. Make a mark on the side of the coverslip to know the side of the cell cover.
2. The concentration of the primary antibody needs to be determined experimentally.
3. If a 22×22 mm coverslip is used in the fourth step, add 30 ul of diluted antibody to the coverslip, and place the coverslip on the glass slide, then place the slides in the wet box. , incubate at room temperature
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