Rabbit UltraAvidin™ Fluorescein Staining Kit

The immunofluorescent staining method is preferred to distinguish fine subcellular structures in fixed tissue sections. The avidin-biotin system for immunostaining has increased the sensitivity for detecting very small amounts of antigen and definitive negative controls can be used as compared to the direct method of labelling.1-3 In this method, the secondary antibody directed against the immunoglobulin of the species in which the first antibody is raised, is biotin labeled. This is allowed to react with the primary antibody, followed by the addition of the fluorescein-conjugated UltraAvidin forming a complex whereby many fluorescein molecules are indirectly bound to each secondary antibody.4, 5 The avidin has an extraordinary affinity for the small molecule biotin forming a noncovalent bond with a dissociation constant of 10-15M making this system highly stable.5, 6 The bound conjugate is then visualized using a fluorescence microscope. The fluorochrome is excited by application of an appropriate light source, the light emitted being filtered by appropriate filters to remove the excitation wavelength and then led to the observer's eye.1 Fluorescein has a maximum absorbance at 492nm (blue) and an emission maximum at 518nm (green). It is covalently attached to UltraAvidin and purified to assure an optimal fluorochrome/protein molar ratio.
Leinco's UltraAvidin is a unique form of avidin, modified to prevent non-specific binding on cell surfaces either due to lectin-like receptor adherence or electrostatic interactions. The secondary Goat anti-rabbit IgG antibody is a F(ab)2 fragment, eliminating non-specific Fc receptor binding and is especially suitable for immunohistochemical studies. It is affinity isolated using rabbit IgG and is adsorbed against human serum proteins and mouse IgG to reduce cross-reactivity with human or mouse tissue sections. The antibody is then conjugated to biotin at a molar ratio to optimize performance. The kit has been developed for enhanced sensitivity whereby low amounts of antigen may be detected or where lower concentrations of primary antibody may be used.