eBioscience™ IHC/ICC Blocking Buffer - High Protein - FAQs

•需要进行封闭操作以减少由非特异性抗体结合产生的背景荧光。常用的封闭步骤是加入2-5％牛血清白蛋白（fraction V defatted BSA）溶液。另一种方法是采用5-10％二抗种属来源的血清溶液。例如，使用山羊抗小鼠IgG二抗时，样品可以被5-10％正常山羊血清有效封闭。为了进一步减少背景荧光，可以使用Image-iT FX 信号增强剂作为预封闭步骤，减少由偶联物上的染料和细胞组分之间电荷的相互作用引起的非特异性标记。
•如果您使用二抗，确保抗体的种属与样品不同。例如不要对小鼠组织使用抗小鼠二抗。
•滴定测定抗体浓度，使用能获得充足的信号的最低浓度。
•试试荧光标记的一抗，因为它应会降低背景干扰，但这样做也会降低信号强度。

人们常使用与荧光抗体来源于同一种属的血清来进行封闭。如果没有使用二抗，这一成分通常为一抗；但如果使用二抗，这种成分就是二抗的宿主。用于直接标记的小鼠一抗时，Fc封闭试剂可以替代鼠血清。CD16 + CD32抗体（FRC-4G8）（货号MFCR004）是一个靶向免疫球蛋白gamma复合体Fc的低亲和力受体。

原因可能很多，包括封闭不足、一抗或二抗浓度过高或一抗或二抗降解。通过“无一抗”对照加以确定是否是二抗的问题。否则，我们建议你们尝试更严格的封闭或更低浓度的一抗或二抗。

进行细胞和组织标记显微检测的最佳浓度为1-10 µg/mL，进行流式细胞术的最佳浓度为0.2-5 µg/mL。最佳浓度需通过试验确定。

如要封闭细胞或组织，可以使用2-5％的牛血清白蛋白（第五组分，脱脂BSA）或是与二抗宿主种属相匹配的5-10％正常血清。其他选择还包括BSA和血清或其他纯化蛋白的混合物。我们提供一种没有种属特异性的即用型封闭液，即BlockAid封闭液（货号B10710）。对于细胞或组织样品，要避免使用脱脂奶粉作为封闭剂，因为它含有大量的磷蛋白、组蛋白和生物素。

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.

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.

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.

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.

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.