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Why is a blocking buffer needed during immunofluorescence staining?

Why is a blocking buffer needed during immunofluorescence staining?

Blocking. Blocking is an important step for minimizing unspecific binding of the primary antibody within the cell. To achieve this, proteins from Bovine Serum Albumin (BSA), milk powder or serum can be used.

How do you make a blocking buffer for immunofluorescence?

Blocking Buffer: Purchase ready-to-use Immunofluorescence Blocking Buffer (#12411), or prepare a 1X PBS / 5% normal serum / 0.3% Triton™ X-100 buffer by adding 0.5 mL normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425) and 30 µL Triton™ X-100 to 9.5 mL 1X PBS. Store at 4°C.

How do you reduce background stains in immunofluorescence?

To reduce non-specific antibody binding and, in consequence, reduce background signal, it is recommended to use a blocking solution prior to incubation with antibodies. The most effective blocking solution will be that containing serum from the same species in which the secondary antibody was raised.

How do you permeabilize cells for immunofluorescence?


  1. Incubate the samples for 10 min with PBS containing either 0.1–0.25% Triton X-100 (or 100 μM digitonin or 0.5% saponin).
  2. The optimal percentage of Triton X-100 should be determined for each protein of interest.
  3. Wash cells in PBS three times for 5 min.

What is the purpose of blocking solution?

A blocking buffer is a solution of irrelevant protein, mixture of proteins, or other compound that passively adsorbs to all remaining binding surfaces of the plate. The blocking buffer is effective if it improves the sensitivity of an assay by reducing background interference and improving the signal-to-noise ratio.

Is blocking necessary for immunofluorescence?

What is blocking in Immunohistochemistry? Blocking is essential for preventing non-specific binding of antibodies or other reagents to the tissue. Even if the antibody has high specificity towards the target, intermolecular forces can promote non-specific binding to other molecules.

Which BSA is used for blocking?

Bovine serum albumin (BSA) blocking buffer is ideal for saturating excess protein-binding sites on membranes and microplates for Western blotting and ELISA applications, respectively. Typically, 1-3% BSA is sufficient for most applications.

What is normal serum for blocking?

Normal serum at 1-5% (w/v) is a common blocking buffer component, because serum carries antibodies that bind to reactive sites and prevent the nonspecific binding of the secondary antibodies used in the assay.

What is immunocytochemical staining?

Immunocytochemistry (ICC) is a technique for detection and visualization of proteins, or other antigens, in cells using antibodies specifically recognizing the target of interest. In ICC, the staining technique is applied on cultured cells or individual cells that have been isolated from eg.

How do you use immunofluorescence staining?

The first step of an immunofluorescence staining protocol is to fixate the sample. This is usually done by incubating the sample for 10 minutes at room temperature in a 4% formalin solution (in PBS, pH 7.4), which crosslinks the proteins. The sample can also be fixated in 100% chilled methanol or acetone.

What fixative is used for immunofluorescence?

Aldehyde-based fixatives such as formaldehyde, formalin (a mixture of dissolved formaldehyde with a lower percentage of methanol), and glutaraldehyde are most commonly used. For most antibodies, CST recommends fixation with 4% formaldehyde (IF Standard protocol).

Why is blocking necessary?

Blocking is essential for preventing non-specific binding of antibodies or other reagents to the tissue. Consequently, non-specific binding prevents visualization of the antigen-antibody binding of interest.