Recently, our lab has been performing immunoflourescence studies using mouse monoclonal antibodies to detect various antigens in mammalian cells. To detect the mouse primary antibodies, we have been using the Alexa Fluor 488 Signal-Amplification Kit for Mouse Antibodies from Molecular Probes. This kit is simple to use, and the enclosed protocol is very easy to follow. It utilizes two Alexa Fluor 488 conjugated antibodies – the first is an Alexa Fluor 488 conjugated rabbit anti-mouse IgG and the second is a goat anti-rabbit IgG that is also Alexa Fluor 488 conjugated. Both antibodies have been affinity purified and adsorbed against human IgG and the goat anti-rabbit antibody has also been adsorbed against mouse IgG. The staining process is fairly straightforward - after the cells have been incubated with the mouse primary antibody, the Alexa Fluor 488 conjugated rabbit anti-mouse antibody is introduced followed by the Alexa Fluor 488 goat anti-rabbit antibody. Thus the fluorescent signal is increased dramatically allowing for the detection of low levels of antigen.
The Signal Amplification Kit is essentially composed of the two antibodies described above. Both components must be stored at -20 oC and both are very sensitive to light. Also, because they may be sensitive to repeated freeze/thaw cycles, it is a good idea to aliquot the antibodies once the kit is received. According to Molecular Probes, with proper care the kit is good for at least 6 months (we have not tried using a kit that is more than 6 months old).
One of the major benefits to using antibodies conjugated to Alexa Fluor 488 is that it is brighter and more stable (both to photobleaching and to changes in pH) compared to fluorescein. And because the absorption/emission spectra of Alexa Fluor 488 is similar to that of fluorescein, there is no need for special filters (or even to change the settings) on the microscope. Also, even with the increased fluorescence, we did not have any noticeable background problems.
Although this kit can be used for both fluorescence microscopy and flow cytometry, we have only used the kit for the former. We essentially followed the protocol that came with the kit, however we made a few minor modifications that helped us produce optimal results. For instance, we first fixed the cells (on coverslips) with formaldehyde and permeabilized them with Triton X-100 (washing with PBS in between). Then, we used PBS containing 6% BSA as a blocking agent for 1 hour at room temperature. Although this step was not in the protocol, for us it helped reduce nonspecific binding of the primary mouse antibody. After the blocking step, we stained the cells with our primary antibody – the incubation period obviously depended on the specific antibody. The only other changes that we made to the protocol is that we incubated the secondary and tertiary antibodies for 1 hour instead of the 30 minutes indicated in the protocol.
Overall, the Molecular Probes-Alexa Fluor 488 Signal-Amplification Kit for Mouse Antibodies has worked very well for us. The kit is very simple to use and comes with enough reagent to perform many assays with one kit. The Molecular Probes-Alexa Fluor 488 Signal-Amplification Kit for Mouse Antibodies is unique in its utilization of two Alexa Fluor 488 conjugates, allowing for brighter and more stable fluorescence without comprising background problems.
Hee Chul Lee
Graduate Student
Dept. of Biochemistry
NYU School of Medicine