I used a neuron-specific antibody to label my neurons. How can I reduce non-specific antibody binding?
A blocking step should be performed to reduce fluorescence due to non-specific antibody binding. A common blocking step is the addition of a 2-5% solution of bovine serum albumin (fraction V defatted BSA). Another approach employs the addition of a 5-10% solution of serum from the species in which the secondary antibodies were raised. For example, when using goat anti-mouse IgG secondary antibodies, samples may be effectively blocked with 5-10% normal goat serum. To further reduce background fluorescence, the Image-iT FX Signal Enhancer can be included as a pre-blocking step to decrease non-specific labeling due to charge interactions between the dyes on the conjugates and the cellular constituents.
If you are using a secondary antibody make sure that the species of the antibody is not the same as the species of the sample. For example do not use an anti-mouse secondary antibody on mouse tissue.
Titrate the antibody to the lowest concentration you can use and still get adequate signal.
Try using a fluorescently tagged primary antibody because it should give reduced background but be aware this can reduce signal intensity.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
What should I use to block my cells for flow cytometry analysis?
Use serum from the same species as the host species of the secondary antibody for blocking. If the serum is not available, use from 2 to 5% BSA (Fraction V, defatted). If using only a primary antibody, such as directly-labeled mouse primary antibodies, a good blocking reagent is Fc block. CD16 + CD32 Antibody (FRC-4G8) (Cat. No. MFCR004 or MA5-16680) is a low-affinity receptor for the Fc region of immunoglobulin gamma complexes.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
After labeling with my antibody, I am seeing non-specific background binding in my cells or tissue. What could be the cause?
There can be many causes, including insufficient blocking, too high a concentration of the primary or secondary antibody, or degraded primary or secondary antibody. A “no-primary antibody” control can help determine if the secondary antibody is at fault. Otherwise, we recommend trying more stringent blocking or lower concentrations of primary and secondary antibodies.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
What concentration of my antibody should I use for cell analysis?
An optimal concentration may be between 1-10 µg/mL for cell and tissue labeling for microscopy, or 0.2-5 µg/mL for flow cytometry. A range of concentrations should be tested to determine what is optimal.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
I know I need to block my samples against non-specific protein binding of my antibodies, but which blocker should I use?
For blocking cells or tissues you may use 2-5% bovine serum albumin (fraction V, defatted BSA) or 5-10% normal serum of the species matching the host species of the secondary antibody. Other options include a mixture of BSA and serum or other purified proteins. We offer a ready-made blocking solution, BlockAid Blocking Solution (Cat. No. B10710), which is not species-specific. For cell or tissue samples, avoid the use of non-fat dry milk as a blocking agent as it contains a high level of phosphoproteins, histones, and biotin.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
