It is a research based on the principle of antigen and antibody specific binding，making the chromogenic reagent ( fluorescein) color of the labeled antibody by chemical reaction，in order to determine the intracellular antigen (polypeptide and protein)，and make the positioning, qualitative and quantitative analysis.
1 Deparaffinize and rehydrate: incubate sections in 2 cylinders of xylene, 15 min each time. Dehydrate in 2 cylinders of pure ethanol for 5 min, followed by dehydrating in gradient ethanol of 85% and 75% ethanol, respectively, 5 min each time. Wash in distilled water.
2 Antigen retrieval: immerse the slides in EDTA antigen retrieval buffer (pH 8.0) and maintain at a sub-boiling temperature for 8 min, standing for 8 min and then followed by another sub-boiling temperature for 7 min. Be sure to prevent buffer solution evaporate. Let air cooling. Wash three times with PBS (pH 7.4) in shaker, 5 min each time. Use the right antigen retrieval buffer and heat extent according to tissue characteristics.
3 Spontaneous fluorescence quenching: eliminate obvious liquid, mark the objective tissue with marker pen. Add spontaneous fluorescence quenching reagent to incubate for 5 min. Wash in running tap water for 10 min.
4 BSA blocking: add 3% BSA to cover the marked tissue to block non-specific binding for 30 min.
5 Primary antibody: throw away the blocking solution slightly. Incubate slides with primary antibody (diluted with PBS appropriately) overnight at 4 °C , placed in a wet box containing a little water.
6 Secondary antibody: wash slides three times with PBS (pH 7.4) in shaker, 5 min each time. Then throw away liquid slightly. Cover objective tissue with secondary antibody (appropriately respond to primary antibody in species), incubate at room temperature for 50 min in dark condition.
7 DAPI counterstain in nucleus: wash three times with PBS (pH 7.4) in shaker, 5 min each time. Then incubate with DAPI solution at room temperature for 10 min in dark place.
8 Mount: wash three times with PBS (pH 7.4) in a shaker, 5 min each time. Throw away liquid slightly, then coverslip with anti-fade mounting medium.
9 Microscopy detection and collect images by Fluorescent Microscopy. DAPI glows blue by UV excitation wavelength 330-380 nm and emission wavelength 420 nm; FITC glows green by excitation wavelength 465-495 nm and emission wavelength 515-555 nm; CY3 glows red by excitation wavelength 510-560 nm and emission wavelength 590 nm.
Nucleus is blue by labeling with DAPI. Positive cells are green or red according to the fluorescent labels used.