Fluorescent antibodies should be identified prior to use. Identification refers to the medical education network finishing label including the potency and the ratio of fluorescein to protein binding. The antibody titer can be titrated by the agar double diffusion method, and the titer is more than 1:16. The basic method for the determination and calculation of the ratio of fluorescein to protein (F/P) is to dilute the prepared fluorescent antibody to A2801≈1.0, and to measure the specific absorption peak of A280 (protein-specific absorption peak) and labeled fluorescein, respectively. Calculated according to the formula.
The higher the F/P value, the more fluorescein bound on the antibody molecule, and vice versa. Fluorescent antibodies generally used for immobilization of specimens are preferably F/P = 1.5, and F/P = 2.4 for viable cell staining.
The method for determining the working concentration of the antibody is similar to the titration of the enzyme-labeled antibody in the ELISA indirect method. Fluorescent antibodies were diluted from 1:4 to 1:256, and the sections were stained with fluorescent antibodies. The highest dilution of the specific fluorescence and the weak non-specific staining is the fluorescent antibody working concentration.
Preservation of fluorescent antibodies should be taken to prevent antibody inactivation and to prevent fluorescence quenching. It is best to pack in small quantities and freeze at -20 °C, so that it can be placed for 3 to 4 years. It can also be stored for 1 to 2 years at 4 °C.
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