免疫荧光操作步骤及注意事项（Immunofluorescence procedures and precautions）.docVIP

免疫荧光操作步骤及注意事项（Immunofluorescence procedures and precautions） Direct immunofluorescence assay for antigen Basic principle The fluorescein was labeled on the corresponding antibody and reacted directly with the corresponding antigen. The method has the advantages of simple and high specificity, less nonspecific fluorescent staining and relatively large amount of labeled antibody used. Reagents and instruments Phosphate buffered saline (PBS): 0.01mol/L, pH7.4 Fluorescently labeled antibody solution: diluted with 0.01mol/L, pH7.4, and PBS Buffered glycerol: analysis of pure, non fluorescent glycerol 9 + pH9.2 0.2M carbonate buffer, 1 prepared Enamel barrel three (with 0.01mol/L, pH7.4, PBS, 1500ml) There is a cover lining porcelain box (within the shop a layer of gauze pad soaked) fluorescence microscope Slide holder filter paper 37 C temperature box etc.. Experimental procedure 1. drops of 0.01mol/L, pH7.4, PBS on the specimen to be tested, discarded after 10min, so that the sample to maintain a certain humidity. The antibody solution fluorescence labeled 2. drops with appropriate dilution, which completely covers were placed in a covered enamel porcelain box, holding a 30min Fixed time (Ref: 30min). 3. take out the slide and put it on the glass rack. First rinse with 0.01mol/L and pH7.4 PBS, then repeat the 0.01mol/L in sequence, PH7.4 PBS three cylinder immersion, 3-5 min per cylinder, oscillating from time to time. 4. remove the slide with the filter paper to absorb excess moisture, but do not make specimens dry, add a drop of glycerol buffer, to cover glass cover. 5. immediately observed with fluorescence microscopy. The intensity of the specific fluorescence of the specimen can be observed by the addition of +: (-) - (+) fluorescence; fluorescence suspicious extremely weak; (+) fluorescence is weak, but clearly visible; (bright fluorescent; (+ + + + +) --++++) bright fluorescence. The detected specimen specific fluorescence staining intensity was + +, and various